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<t>CD74</t> −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p
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1) Product Images from "CD74 is a regulator of hematopoietic stem cell maintenance"

Article Title: CD74 is a regulator of hematopoietic stem cell maintenance

Journal: PLoS Biology

doi: 10.1371/journal.pbio.3001121

CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, Two Tailed Test

CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, Transplantation Assay, Two Tailed Test

Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Mouse Assay, Derivative Assay, Purification, Expressing, FACS, Two Tailed Test

CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, FACS, Staining, Two Tailed Test

CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *
Figure Legend Snippet: CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *

Techniques Used: Cell Culture, Incubation, Blocking Assay, FACS, Injection, Mouse Assay

Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p
Figure Legend Snippet: Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p

Techniques Used: FACS, Expressing, Mouse Assay, Injection, Staining, BrdU Incorporation Assay, Two Tailed Test

CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *
Figure Legend Snippet: CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *

Techniques Used: Irradiation, Mouse Assay, Two Tailed Test

CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Expressing, FACS, Staining, Mouse Assay, Binding Assay, Chromatin Immunoprecipitation, Cell Culture, Two Tailed Test

2) Product Images from "CD74 is a regulator of hematopoietic stem cell maintenance"

Article Title: CD74 is a regulator of hematopoietic stem cell maintenance

Journal: PLoS Biology

doi: 10.1371/journal.pbio.3001121

CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, Two Tailed Test

CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, Transplantation Assay, Two Tailed Test

Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Mouse Assay, Derivative Assay, Purification, Expressing, FACS, Two Tailed Test

CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, FACS, Staining, Two Tailed Test

CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *
Figure Legend Snippet: CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *

Techniques Used: Cell Culture, Incubation, Blocking Assay, FACS, Injection, Mouse Assay

Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p
Figure Legend Snippet: Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p

Techniques Used: FACS, Expressing, Mouse Assay, Injection, Staining, BrdU Incorporation Assay, Two Tailed Test

CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *
Figure Legend Snippet: CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *

Techniques Used: Irradiation, Mouse Assay, Two Tailed Test

CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Expressing, FACS, Staining, Mouse Assay, Binding Assay, Chromatin Immunoprecipitation, Cell Culture, Two Tailed Test

3) Product Images from "CD74 is a regulator of hematopoietic stem cell maintenance"

Article Title: CD74 is a regulator of hematopoietic stem cell maintenance

Journal: PLoS Biology

doi: 10.1371/journal.pbio.3001121

CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, Two Tailed Test

CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, Transplantation Assay, Two Tailed Test

Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Mouse Assay, Derivative Assay, Purification, Expressing, FACS, Two Tailed Test

CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, FACS, Staining, Two Tailed Test

CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *
Figure Legend Snippet: CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *

Techniques Used: Cell Culture, Incubation, Blocking Assay, FACS, Injection, Mouse Assay

Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p
Figure Legend Snippet: Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p

Techniques Used: FACS, Expressing, Mouse Assay, Injection, Staining, BrdU Incorporation Assay, Two Tailed Test

CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *
Figure Legend Snippet: CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *

Techniques Used: Irradiation, Mouse Assay, Two Tailed Test

CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Expressing, FACS, Staining, Mouse Assay, Binding Assay, Chromatin Immunoprecipitation, Cell Culture, Two Tailed Test

4) Product Images from "CD74 is a regulator of hematopoietic stem cell maintenance"

Article Title: CD74 is a regulator of hematopoietic stem cell maintenance

Journal: PLoS Biology

doi: 10.1371/journal.pbio.3001121

CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, Two Tailed Test

CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, Transplantation Assay, Two Tailed Test

Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Mouse Assay, Derivative Assay, Purification, Expressing, FACS, Two Tailed Test

CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, FACS, Staining, Two Tailed Test

CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *
Figure Legend Snippet: CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *

Techniques Used: Cell Culture, Incubation, Blocking Assay, FACS, Injection, Mouse Assay

Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p
Figure Legend Snippet: Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p

Techniques Used: FACS, Expressing, Mouse Assay, Injection, Staining, BrdU Incorporation Assay, Two Tailed Test

CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *
Figure Legend Snippet: CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *

Techniques Used: Irradiation, Mouse Assay, Two Tailed Test

CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Expressing, FACS, Staining, Mouse Assay, Binding Assay, Chromatin Immunoprecipitation, Cell Culture, Two Tailed Test

5) Product Images from "CD74 is a regulator of hematopoietic stem cell maintenance"

Article Title: CD74 is a regulator of hematopoietic stem cell maintenance

Journal: PLoS Biology

doi: 10.1371/journal.pbio.3001121

CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, Two Tailed Test

CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, Transplantation Assay, Two Tailed Test

Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Mouse Assay, Derivative Assay, Purification, Expressing, FACS, Two Tailed Test

CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, FACS, Staining, Two Tailed Test

CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *
Figure Legend Snippet: CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *

Techniques Used: Cell Culture, Incubation, Blocking Assay, FACS, Injection, Mouse Assay

Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p
Figure Legend Snippet: Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p

Techniques Used: FACS, Expressing, Mouse Assay, Injection, Staining, BrdU Incorporation Assay, Two Tailed Test

CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *
Figure Legend Snippet: CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *

Techniques Used: Irradiation, Mouse Assay, Two Tailed Test

CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Expressing, FACS, Staining, Mouse Assay, Binding Assay, Chromatin Immunoprecipitation, Cell Culture, Two Tailed Test

6) Product Images from "CD74 is a regulator of hematopoietic stem cell maintenance"

Article Title: CD74 is a regulator of hematopoietic stem cell maintenance

Journal: PLoS Biology

doi: 10.1371/journal.pbio.3001121

CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, Two Tailed Test

CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, Transplantation Assay, Two Tailed Test

Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Mouse Assay, Derivative Assay, Purification, Expressing, FACS, Two Tailed Test

CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, FACS, Staining, Two Tailed Test

CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *
Figure Legend Snippet: CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *

Techniques Used: Cell Culture, Incubation, Blocking Assay, FACS, Injection, Mouse Assay

Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p
Figure Legend Snippet: Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p

Techniques Used: FACS, Expressing, Mouse Assay, Injection, Staining, BrdU Incorporation Assay, Two Tailed Test

CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *
Figure Legend Snippet: CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *

Techniques Used: Irradiation, Mouse Assay, Two Tailed Test

CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Expressing, FACS, Staining, Mouse Assay, Binding Assay, Chromatin Immunoprecipitation, Cell Culture, Two Tailed Test

7) Product Images from "CD74 is a regulator of hematopoietic stem cell maintenance"

Article Title: CD74 is a regulator of hematopoietic stem cell maintenance

Journal: PLoS Biology

doi: 10.1371/journal.pbio.3001121

CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, Two Tailed Test

CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, Transplantation Assay, Two Tailed Test

Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Mouse Assay, Derivative Assay, Purification, Expressing, FACS, Two Tailed Test

CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, FACS, Staining, Two Tailed Test

CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *
Figure Legend Snippet: CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *

Techniques Used: Cell Culture, Incubation, Blocking Assay, FACS, Injection, Mouse Assay

Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p
Figure Legend Snippet: Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p

Techniques Used: FACS, Expressing, Mouse Assay, Injection, Staining, BrdU Incorporation Assay, Two Tailed Test

CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *
Figure Legend Snippet: CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *

Techniques Used: Irradiation, Mouse Assay, Two Tailed Test

CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Expressing, FACS, Staining, Mouse Assay, Binding Assay, Chromatin Immunoprecipitation, Cell Culture, Two Tailed Test

8) Product Images from "CD74 is a regulator of hematopoietic stem cell maintenance"

Article Title: CD74 is a regulator of hematopoietic stem cell maintenance

Journal: PLoS Biology

doi: 10.1371/journal.pbio.3001121

CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, Two Tailed Test

CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, Transplantation Assay, Two Tailed Test

Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Mouse Assay, Derivative Assay, Purification, Expressing, FACS, Two Tailed Test

CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, FACS, Staining, Two Tailed Test

CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *
Figure Legend Snippet: CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *

Techniques Used: Cell Culture, Incubation, Blocking Assay, FACS, Injection, Mouse Assay

Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p
Figure Legend Snippet: Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p

Techniques Used: FACS, Expressing, Mouse Assay, Injection, Staining, BrdU Incorporation Assay, Two Tailed Test

CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *
Figure Legend Snippet: CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *

Techniques Used: Irradiation, Mouse Assay, Two Tailed Test

CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Expressing, FACS, Staining, Mouse Assay, Binding Assay, Chromatin Immunoprecipitation, Cell Culture, Two Tailed Test

9) Product Images from "CD74 is a regulator of hematopoietic stem cell maintenance"

Article Title: CD74 is a regulator of hematopoietic stem cell maintenance

Journal: PLoS Biology

doi: 10.1371/journal.pbio.3001121

CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, Two Tailed Test

CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, Transplantation Assay, Two Tailed Test

Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Mouse Assay, Derivative Assay, Purification, Expressing, FACS, Two Tailed Test

CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, FACS, Staining, Two Tailed Test

CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *
Figure Legend Snippet: CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *

Techniques Used: Cell Culture, Incubation, Blocking Assay, FACS, Injection, Mouse Assay

Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p
Figure Legend Snippet: Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p

Techniques Used: FACS, Expressing, Mouse Assay, Injection, Staining, BrdU Incorporation Assay, Two Tailed Test

CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *
Figure Legend Snippet: CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *

Techniques Used: Irradiation, Mouse Assay, Two Tailed Test

CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Expressing, FACS, Staining, Mouse Assay, Binding Assay, Chromatin Immunoprecipitation, Cell Culture, Two Tailed Test

10) Product Images from "CD74 is a regulator of hematopoietic stem cell maintenance"

Article Title: CD74 is a regulator of hematopoietic stem cell maintenance

Journal: PLoS Biology

doi: 10.1371/journal.pbio.3001121

CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, Two Tailed Test

CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, Transplantation Assay, Two Tailed Test

Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Mouse Assay, Derivative Assay, Purification, Expressing, FACS, Two Tailed Test

CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, FACS, Staining, Two Tailed Test

CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *
Figure Legend Snippet: CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *

Techniques Used: Cell Culture, Incubation, Blocking Assay, FACS, Injection, Mouse Assay

Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p
Figure Legend Snippet: Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p

Techniques Used: FACS, Expressing, Mouse Assay, Injection, Staining, BrdU Incorporation Assay, Two Tailed Test

CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *
Figure Legend Snippet: CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *

Techniques Used: Irradiation, Mouse Assay, Two Tailed Test

CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Expressing, FACS, Staining, Mouse Assay, Binding Assay, Chromatin Immunoprecipitation, Cell Culture, Two Tailed Test

11) Product Images from "CD74 is a regulator of hematopoietic stem cell maintenance"

Article Title: CD74 is a regulator of hematopoietic stem cell maintenance

Journal: PLoS Biology

doi: 10.1371/journal.pbio.3001121

CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, Two Tailed Test

CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, Transplantation Assay, Two Tailed Test

Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Mouse Assay, Derivative Assay, Purification, Expressing, FACS, Two Tailed Test

CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, FACS, Staining, Two Tailed Test

CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *
Figure Legend Snippet: CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *

Techniques Used: Cell Culture, Incubation, Blocking Assay, FACS, Injection, Mouse Assay

Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p
Figure Legend Snippet: Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p

Techniques Used: FACS, Expressing, Mouse Assay, Injection, Staining, BrdU Incorporation Assay, Two Tailed Test

CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *
Figure Legend Snippet: CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *

Techniques Used: Irradiation, Mouse Assay, Two Tailed Test

CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Expressing, FACS, Staining, Mouse Assay, Binding Assay, Chromatin Immunoprecipitation, Cell Culture, Two Tailed Test

12) Product Images from "CD74 is a regulator of hematopoietic stem cell maintenance"

Article Title: CD74 is a regulator of hematopoietic stem cell maintenance

Journal: PLoS Biology

doi: 10.1371/journal.pbio.3001121

CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, Two Tailed Test

CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, Transplantation Assay, Two Tailed Test

Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Mouse Assay, Derivative Assay, Purification, Expressing, FACS, Two Tailed Test

CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, FACS, Staining, Two Tailed Test

CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *
Figure Legend Snippet: CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *

Techniques Used: Cell Culture, Incubation, Blocking Assay, FACS, Injection, Mouse Assay

Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p
Figure Legend Snippet: Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p

Techniques Used: FACS, Expressing, Mouse Assay, Injection, Staining, BrdU Incorporation Assay, Two Tailed Test

CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *
Figure Legend Snippet: CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *

Techniques Used: Irradiation, Mouse Assay, Two Tailed Test

CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Expressing, FACS, Staining, Mouse Assay, Binding Assay, Chromatin Immunoprecipitation, Cell Culture, Two Tailed Test

13) Product Images from "17?-Estradiol Inhibits Wound Healing in Male Mice via Estrogen Receptor-?"

Article Title: 17?-Estradiol Inhibits Wound Healing in Male Mice via Estrogen Receptor-?

Journal: The American Journal of Pathology

doi: 10.2353/ajpath.2010.090432

Effects of chronic 17β-estradiol on wound inflammation in castrated mice. A: Immunostaining for the neutrophil marker Ly6G, and overall neutrophil numbers, in day three wounds from castrated (CSX, CX) MF1 mice, some treated with 17β-estradiol (E2) from days −14 to +3 (CSX +E2, CXE). B: Immunostaining for the macrophage marker Mac3, and numbers of macrophages, in day three wounds from E2-treated CSX MF1 mice. C: Immunostaining for arginase 1, a marker of AA macrophages, and day three wound numbers of arginase 1-positive cells, in E2-treated CSX MF1 mice. Immunostaining for TNF-α ( D ), MIF ( E ), and CD74 ( F ) in day three wounds from E2-treated CSX mice. G: Day three wound expression of the Tnfa , Mif , and Cd74 genes, determined by qPCR. H: Cellular levels of CD74 protein in peritoneal macrophages activated with LPS (+) for 2 hours (Con) and then treated with 100 nmol/L E2 or left untreated (Con) for a further three hours (immunoblotting). I: Expression of the Cd74 gene in control and 100 nmol/L E2-treated macrophages (qPCR). Statistical significance ( C and F ) was determined by unpaired Student’s t -tests: * P
Figure Legend Snippet: Effects of chronic 17β-estradiol on wound inflammation in castrated mice. A: Immunostaining for the neutrophil marker Ly6G, and overall neutrophil numbers, in day three wounds from castrated (CSX, CX) MF1 mice, some treated with 17β-estradiol (E2) from days −14 to +3 (CSX +E2, CXE). B: Immunostaining for the macrophage marker Mac3, and numbers of macrophages, in day three wounds from E2-treated CSX MF1 mice. C: Immunostaining for arginase 1, a marker of AA macrophages, and day three wound numbers of arginase 1-positive cells, in E2-treated CSX MF1 mice. Immunostaining for TNF-α ( D ), MIF ( E ), and CD74 ( F ) in day three wounds from E2-treated CSX mice. G: Day three wound expression of the Tnfa , Mif , and Cd74 genes, determined by qPCR. H: Cellular levels of CD74 protein in peritoneal macrophages activated with LPS (+) for 2 hours (Con) and then treated with 100 nmol/L E2 or left untreated (Con) for a further three hours (immunoblotting). I: Expression of the Cd74 gene in control and 100 nmol/L E2-treated macrophages (qPCR). Statistical significance ( C and F ) was determined by unpaired Student’s t -tests: * P

Techniques Used: Mouse Assay, Immunostaining, Marker, Expressing, Real-time Polymerase Chain Reaction

14) Product Images from "CD74 is a regulator of hematopoietic stem cell maintenance"

Article Title: CD74 is a regulator of hematopoietic stem cell maintenance

Journal: PLoS Biology

doi: 10.1371/journal.pbio.3001121

CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, Two Tailed Test

CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, Transplantation Assay, Two Tailed Test

Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Mouse Assay, Derivative Assay, Purification, Expressing, FACS, Two Tailed Test

CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, FACS, Staining, Two Tailed Test

CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *
Figure Legend Snippet: CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *

Techniques Used: Cell Culture, Incubation, Blocking Assay, FACS, Injection, Mouse Assay

Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p
Figure Legend Snippet: Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p

Techniques Used: FACS, Expressing, Mouse Assay, Injection, Staining, BrdU Incorporation Assay, Two Tailed Test

CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *
Figure Legend Snippet: CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *

Techniques Used: Irradiation, Mouse Assay, Two Tailed Test

CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Expressing, FACS, Staining, Mouse Assay, Binding Assay, Chromatin Immunoprecipitation, Cell Culture, Two Tailed Test

15) Product Images from "CD74 is a regulator of hematopoietic stem cell maintenance"

Article Title: CD74 is a regulator of hematopoietic stem cell maintenance

Journal: PLoS Biology

doi: 10.1371/journal.pbio.3001121

CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, Two Tailed Test

CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, Transplantation Assay, Two Tailed Test

Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Mouse Assay, Derivative Assay, Purification, Expressing, FACS, Two Tailed Test

CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, FACS, Staining, Two Tailed Test

CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *
Figure Legend Snippet: CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *

Techniques Used: Cell Culture, Incubation, Blocking Assay, FACS, Injection, Mouse Assay

Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p
Figure Legend Snippet: Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p

Techniques Used: FACS, Expressing, Mouse Assay, Injection, Staining, BrdU Incorporation Assay, Two Tailed Test

CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *
Figure Legend Snippet: CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *

Techniques Used: Irradiation, Mouse Assay, Two Tailed Test

CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Expressing, FACS, Staining, Mouse Assay, Binding Assay, Chromatin Immunoprecipitation, Cell Culture, Two Tailed Test

16) Product Images from "CD74 is a regulator of hematopoietic stem cell maintenance"

Article Title: CD74 is a regulator of hematopoietic stem cell maintenance

Journal: PLoS Biology

doi: 10.1371/journal.pbio.3001121

CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, Two Tailed Test

CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, Transplantation Assay, Two Tailed Test

Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Mouse Assay, Derivative Assay, Purification, Expressing, FACS, Two Tailed Test

CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, FACS, Staining, Two Tailed Test

CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *
Figure Legend Snippet: CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *

Techniques Used: Cell Culture, Incubation, Blocking Assay, FACS, Injection, Mouse Assay

Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p
Figure Legend Snippet: Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p

Techniques Used: FACS, Expressing, Mouse Assay, Injection, Staining, BrdU Incorporation Assay, Two Tailed Test

CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *
Figure Legend Snippet: CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *

Techniques Used: Irradiation, Mouse Assay, Two Tailed Test

CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Expressing, FACS, Staining, Mouse Assay, Binding Assay, Chromatin Immunoprecipitation, Cell Culture, Two Tailed Test

17) Product Images from "CD74 is a regulator of hematopoietic stem cell maintenance"

Article Title: CD74 is a regulator of hematopoietic stem cell maintenance

Journal: PLoS Biology

doi: 10.1371/journal.pbio.3001121

CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, Two Tailed Test

CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, Transplantation Assay, Two Tailed Test

Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Mouse Assay, Derivative Assay, Purification, Expressing, FACS, Two Tailed Test

CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, FACS, Staining, Two Tailed Test

CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *
Figure Legend Snippet: CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *

Techniques Used: Cell Culture, Incubation, Blocking Assay, FACS, Injection, Mouse Assay

Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p
Figure Legend Snippet: Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p

Techniques Used: FACS, Expressing, Mouse Assay, Injection, Staining, BrdU Incorporation Assay, Two Tailed Test

CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *
Figure Legend Snippet: CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *

Techniques Used: Irradiation, Mouse Assay, Two Tailed Test

CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Expressing, FACS, Staining, Mouse Assay, Binding Assay, Chromatin Immunoprecipitation, Cell Culture, Two Tailed Test

18) Product Images from "CD74 is a regulator of hematopoietic stem cell maintenance"

Article Title: CD74 is a regulator of hematopoietic stem cell maintenance

Journal: PLoS Biology

doi: 10.1371/journal.pbio.3001121

CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, Two Tailed Test

CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, Transplantation Assay, Two Tailed Test

Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Mouse Assay, Derivative Assay, Purification, Expressing, FACS, Two Tailed Test

CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, FACS, Staining, Two Tailed Test

CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *
Figure Legend Snippet: CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *

Techniques Used: Cell Culture, Incubation, Blocking Assay, FACS, Injection, Mouse Assay

Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p
Figure Legend Snippet: Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p

Techniques Used: FACS, Expressing, Mouse Assay, Injection, Staining, BrdU Incorporation Assay, Two Tailed Test

CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *
Figure Legend Snippet: CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *

Techniques Used: Irradiation, Mouse Assay, Two Tailed Test

CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Expressing, FACS, Staining, Mouse Assay, Binding Assay, Chromatin Immunoprecipitation, Cell Culture, Two Tailed Test

19) Product Images from "CD74 is a regulator of hematopoietic stem cell maintenance"

Article Title: CD74 is a regulator of hematopoietic stem cell maintenance

Journal: PLoS Biology

doi: 10.1371/journal.pbio.3001121

CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, Two Tailed Test

CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, Transplantation Assay, Two Tailed Test

Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Mouse Assay, Derivative Assay, Purification, Expressing, FACS, Two Tailed Test

CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, FACS, Staining, Two Tailed Test

CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *
Figure Legend Snippet: CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *

Techniques Used: Cell Culture, Incubation, Blocking Assay, FACS, Injection, Mouse Assay

Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p
Figure Legend Snippet: Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p

Techniques Used: FACS, Expressing, Mouse Assay, Injection, Staining, BrdU Incorporation Assay, Two Tailed Test

CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *
Figure Legend Snippet: CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *

Techniques Used: Irradiation, Mouse Assay, Two Tailed Test

CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Expressing, FACS, Staining, Mouse Assay, Binding Assay, Chromatin Immunoprecipitation, Cell Culture, Two Tailed Test

20) Product Images from "CD74 is a regulator of hematopoietic stem cell maintenance"

Article Title: CD74 is a regulator of hematopoietic stem cell maintenance

Journal: PLoS Biology

doi: 10.1371/journal.pbio.3001121

CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, Two Tailed Test

CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, Transplantation Assay, Two Tailed Test

Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Mouse Assay, Derivative Assay, Purification, Expressing, FACS, Two Tailed Test

CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, FACS, Staining, Two Tailed Test

CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *
Figure Legend Snippet: CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *

Techniques Used: Cell Culture, Incubation, Blocking Assay, FACS, Injection, Mouse Assay

Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p
Figure Legend Snippet: Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p

Techniques Used: FACS, Expressing, Mouse Assay, Injection, Staining, BrdU Incorporation Assay, Two Tailed Test

CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *
Figure Legend Snippet: CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *

Techniques Used: Irradiation, Mouse Assay, Two Tailed Test

CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Expressing, FACS, Staining, Mouse Assay, Binding Assay, Chromatin Immunoprecipitation, Cell Culture, Two Tailed Test

21) Product Images from "Lipidated promiscuous peptide augments the expression of MHC-II molecules on dendritic cells and activates T cells"

Article Title: Lipidated promiscuous peptide augments the expression of MHC-II molecules on dendritic cells and activates T cells

Journal: The Indian Journal of Medical Research

doi:

L91 induces MHC-II but downregulates CD74 expression. Bone marrow derived cells (BMDCs) were cultured in medium containing L91, F91 or lipopolysaccharides (LPS). The CD11c + cells were analyzed by flowcytometry for the expression of (a) MHC-II; (b) CD74 (immature MHC-II). Numbers in the flow cytometry histograms indicate mean fluorescence intensity (MFI) and are representative of 3 independent experiments; (c) bar graphs depict the change in the ratio of MHC-II to CD74. The cells cultured with medium alone and with LPS were used as negative and positive controls, respectively. Data are shown as the mean±SD are from 3 independent experiments. *** P
Figure Legend Snippet: L91 induces MHC-II but downregulates CD74 expression. Bone marrow derived cells (BMDCs) were cultured in medium containing L91, F91 or lipopolysaccharides (LPS). The CD11c + cells were analyzed by flowcytometry for the expression of (a) MHC-II; (b) CD74 (immature MHC-II). Numbers in the flow cytometry histograms indicate mean fluorescence intensity (MFI) and are representative of 3 independent experiments; (c) bar graphs depict the change in the ratio of MHC-II to CD74. The cells cultured with medium alone and with LPS were used as negative and positive controls, respectively. Data are shown as the mean±SD are from 3 independent experiments. *** P

Techniques Used: Expressing, Derivative Assay, Cell Culture, Flow Cytometry, Cytometry, Fluorescence

22) Product Images from "CD74 is a regulator of hematopoietic stem cell maintenance"

Article Title: CD74 is a regulator of hematopoietic stem cell maintenance

Journal: PLoS Biology

doi: 10.1371/journal.pbio.3001121

CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, Two Tailed Test

CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, Transplantation Assay, Two Tailed Test

Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Mouse Assay, Derivative Assay, Purification, Expressing, FACS, Two Tailed Test

CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, FACS, Staining, Two Tailed Test

CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *
Figure Legend Snippet: CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *

Techniques Used: Cell Culture, Incubation, Blocking Assay, FACS, Injection, Mouse Assay

Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p
Figure Legend Snippet: Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p

Techniques Used: FACS, Expressing, Mouse Assay, Injection, Staining, BrdU Incorporation Assay, Two Tailed Test

CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *
Figure Legend Snippet: CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *

Techniques Used: Irradiation, Mouse Assay, Two Tailed Test

CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Expressing, FACS, Staining, Mouse Assay, Binding Assay, Chromatin Immunoprecipitation, Cell Culture, Two Tailed Test

23) Product Images from "CD74 is a regulator of hematopoietic stem cell maintenance"

Article Title: CD74 is a regulator of hematopoietic stem cell maintenance

Journal: PLoS Biology

doi: 10.1371/journal.pbio.3001121

CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, Two Tailed Test

CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, Transplantation Assay, Two Tailed Test

Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Mouse Assay, Derivative Assay, Purification, Expressing, FACS, Two Tailed Test

CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, FACS, Staining, Two Tailed Test

CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *
Figure Legend Snippet: CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *

Techniques Used: Cell Culture, Incubation, Blocking Assay, FACS, Injection, Mouse Assay

Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p
Figure Legend Snippet: Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p

Techniques Used: FACS, Expressing, Mouse Assay, Injection, Staining, BrdU Incorporation Assay, Two Tailed Test

CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *
Figure Legend Snippet: CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *

Techniques Used: Irradiation, Mouse Assay, Two Tailed Test

CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Expressing, FACS, Staining, Mouse Assay, Binding Assay, Chromatin Immunoprecipitation, Cell Culture, Two Tailed Test

24) Product Images from "CD74 is a regulator of hematopoietic stem cell maintenance"

Article Title: CD74 is a regulator of hematopoietic stem cell maintenance

Journal: PLoS Biology

doi: 10.1371/journal.pbio.3001121

CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, Two Tailed Test

CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, Transplantation Assay, Two Tailed Test

Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Mouse Assay, Derivative Assay, Purification, Expressing, FACS, Two Tailed Test

CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, FACS, Staining, Two Tailed Test

CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *
Figure Legend Snippet: CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *

Techniques Used: Cell Culture, Incubation, Blocking Assay, FACS, Injection, Mouse Assay

Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p
Figure Legend Snippet: Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p

Techniques Used: FACS, Expressing, Mouse Assay, Injection, Staining, BrdU Incorporation Assay, Two Tailed Test

CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *
Figure Legend Snippet: CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *

Techniques Used: Irradiation, Mouse Assay, Two Tailed Test

CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Expressing, FACS, Staining, Mouse Assay, Binding Assay, Chromatin Immunoprecipitation, Cell Culture, Two Tailed Test

25) Product Images from "CD74 is a regulator of hematopoietic stem cell maintenance"

Article Title: CD74 is a regulator of hematopoietic stem cell maintenance

Journal: PLoS Biology

doi: 10.1371/journal.pbio.3001121

CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, Two Tailed Test

CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, Transplantation Assay, Two Tailed Test

Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Mouse Assay, Derivative Assay, Purification, Expressing, FACS, Two Tailed Test

CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, FACS, Staining, Two Tailed Test

CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *
Figure Legend Snippet: CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *

Techniques Used: Cell Culture, Incubation, Blocking Assay, FACS, Injection, Mouse Assay

Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p
Figure Legend Snippet: Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p

Techniques Used: FACS, Expressing, Mouse Assay, Injection, Staining, BrdU Incorporation Assay, Two Tailed Test

CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *
Figure Legend Snippet: CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *

Techniques Used: Irradiation, Mouse Assay, Two Tailed Test

CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Expressing, FACS, Staining, Mouse Assay, Binding Assay, Chromatin Immunoprecipitation, Cell Culture, Two Tailed Test

26) Product Images from "The asparaginyl endopeptidase legumain after experimental stroke"

Article Title: The asparaginyl endopeptidase legumain after experimental stroke

Journal: Journal of Cerebral Blood Flow & Metabolism

doi: 10.1038/jcbfm.2010.39

Analysis of CD74 positive cells in the ischemic hemisphere of legumain (lgmn) wild-type and deficient mice after transient middle cerebral artery occlusion (tMCAO). Quantification of CD74 + cells from the ischemic hemisphere of lgmn wild-type (
Figure Legend Snippet: Analysis of CD74 positive cells in the ischemic hemisphere of legumain (lgmn) wild-type and deficient mice after transient middle cerebral artery occlusion (tMCAO). Quantification of CD74 + cells from the ischemic hemisphere of lgmn wild-type (

Techniques Used: Mouse Assay

27) Product Images from "CD74 is a regulator of hematopoietic stem cell maintenance"

Article Title: CD74 is a regulator of hematopoietic stem cell maintenance

Journal: PLoS Biology

doi: 10.1371/journal.pbio.3001121

CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, Two Tailed Test

CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, Transplantation Assay, Two Tailed Test

Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Mouse Assay, Derivative Assay, Purification, Expressing, FACS, Two Tailed Test

CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, FACS, Staining, Two Tailed Test

CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *
Figure Legend Snippet: CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *

Techniques Used: Cell Culture, Incubation, Blocking Assay, FACS, Injection, Mouse Assay

Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p
Figure Legend Snippet: Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p

Techniques Used: FACS, Expressing, Mouse Assay, Injection, Staining, BrdU Incorporation Assay, Two Tailed Test

CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *
Figure Legend Snippet: CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *

Techniques Used: Irradiation, Mouse Assay, Two Tailed Test

CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Expressing, FACS, Staining, Mouse Assay, Binding Assay, Chromatin Immunoprecipitation, Cell Culture, Two Tailed Test

28) Product Images from "CD74 is a regulator of hematopoietic stem cell maintenance"

Article Title: CD74 is a regulator of hematopoietic stem cell maintenance

Journal: PLoS Biology

doi: 10.1371/journal.pbio.3001121

CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, Two Tailed Test

CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, Transplantation Assay, Two Tailed Test

Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Mouse Assay, Derivative Assay, Purification, Expressing, FACS, Two Tailed Test

CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, FACS, Staining, Two Tailed Test

CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *
Figure Legend Snippet: CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *

Techniques Used: Cell Culture, Incubation, Blocking Assay, FACS, Injection, Mouse Assay

Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p
Figure Legend Snippet: Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p

Techniques Used: FACS, Expressing, Mouse Assay, Injection, Staining, BrdU Incorporation Assay, Two Tailed Test

CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *
Figure Legend Snippet: CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *

Techniques Used: Irradiation, Mouse Assay, Two Tailed Test

CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Expressing, FACS, Staining, Mouse Assay, Binding Assay, Chromatin Immunoprecipitation, Cell Culture, Two Tailed Test

29) Product Images from "CD74 is a regulator of hematopoietic stem cell maintenance"

Article Title: CD74 is a regulator of hematopoietic stem cell maintenance

Journal: PLoS Biology

doi: 10.1371/journal.pbio.3001121

CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, Two Tailed Test

CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, Transplantation Assay, Two Tailed Test

Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Mouse Assay, Derivative Assay, Purification, Expressing, FACS, Two Tailed Test

CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, FACS, Staining, Two Tailed Test

CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *
Figure Legend Snippet: CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *

Techniques Used: Cell Culture, Incubation, Blocking Assay, FACS, Injection, Mouse Assay

Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p
Figure Legend Snippet: Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p

Techniques Used: FACS, Expressing, Mouse Assay, Injection, Staining, BrdU Incorporation Assay, Two Tailed Test

CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *
Figure Legend Snippet: CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *

Techniques Used: Irradiation, Mouse Assay, Two Tailed Test

CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Expressing, FACS, Staining, Mouse Assay, Binding Assay, Chromatin Immunoprecipitation, Cell Culture, Two Tailed Test

30) Product Images from "CD74 is a regulator of hematopoietic stem cell maintenance"

Article Title: CD74 is a regulator of hematopoietic stem cell maintenance

Journal: PLoS Biology

doi: 10.1371/journal.pbio.3001121

CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, Two Tailed Test

CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, Transplantation Assay, Two Tailed Test

Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Mouse Assay, Derivative Assay, Purification, Expressing, FACS, Two Tailed Test

CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, FACS, Staining, Two Tailed Test

CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *
Figure Legend Snippet: CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *

Techniques Used: Cell Culture, Incubation, Blocking Assay, FACS, Injection, Mouse Assay

Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p
Figure Legend Snippet: Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p

Techniques Used: FACS, Expressing, Mouse Assay, Injection, Staining, BrdU Incorporation Assay, Two Tailed Test

CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *
Figure Legend Snippet: CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *

Techniques Used: Irradiation, Mouse Assay, Two Tailed Test

CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Expressing, FACS, Staining, Mouse Assay, Binding Assay, Chromatin Immunoprecipitation, Cell Culture, Two Tailed Test

31) Product Images from "CD74 is a regulator of hematopoietic stem cell maintenance"

Article Title: CD74 is a regulator of hematopoietic stem cell maintenance

Journal: PLoS Biology

doi: 10.1371/journal.pbio.3001121

CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, Two Tailed Test

CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, Transplantation Assay, Two Tailed Test

Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Mouse Assay, Derivative Assay, Purification, Expressing, FACS, Two Tailed Test

CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, FACS, Staining, Two Tailed Test

CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *
Figure Legend Snippet: CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *

Techniques Used: Cell Culture, Incubation, Blocking Assay, FACS, Injection, Mouse Assay

Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p
Figure Legend Snippet: Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p

Techniques Used: FACS, Expressing, Mouse Assay, Injection, Staining, BrdU Incorporation Assay, Two Tailed Test

CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *
Figure Legend Snippet: CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *

Techniques Used: Irradiation, Mouse Assay, Two Tailed Test

CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Expressing, FACS, Staining, Mouse Assay, Binding Assay, Chromatin Immunoprecipitation, Cell Culture, Two Tailed Test

32) Product Images from "CD74 is a regulator of hematopoietic stem cell maintenance"

Article Title: CD74 is a regulator of hematopoietic stem cell maintenance

Journal: PLoS Biology

doi: 10.1371/journal.pbio.3001121

CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, Two Tailed Test

CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, Transplantation Assay, Two Tailed Test

Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Mouse Assay, Derivative Assay, Purification, Expressing, FACS, Two Tailed Test

CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, FACS, Staining, Two Tailed Test

CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *
Figure Legend Snippet: CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *

Techniques Used: Cell Culture, Incubation, Blocking Assay, FACS, Injection, Mouse Assay

Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p
Figure Legend Snippet: Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p

Techniques Used: FACS, Expressing, Mouse Assay, Injection, Staining, BrdU Incorporation Assay, Two Tailed Test

CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *
Figure Legend Snippet: CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *

Techniques Used: Irradiation, Mouse Assay, Two Tailed Test

CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Expressing, FACS, Staining, Mouse Assay, Binding Assay, Chromatin Immunoprecipitation, Cell Culture, Two Tailed Test

33) Product Images from "CD74 is a regulator of hematopoietic stem cell maintenance"

Article Title: CD74 is a regulator of hematopoietic stem cell maintenance

Journal: PLoS Biology

doi: 10.1371/journal.pbio.3001121

CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, Two Tailed Test

CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, Transplantation Assay, Two Tailed Test

Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Mouse Assay, Derivative Assay, Purification, Expressing, FACS, Two Tailed Test

CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, FACS, Staining, Two Tailed Test

CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *
Figure Legend Snippet: CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *

Techniques Used: Cell Culture, Incubation, Blocking Assay, FACS, Injection, Mouse Assay

Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p
Figure Legend Snippet: Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p

Techniques Used: FACS, Expressing, Mouse Assay, Injection, Staining, BrdU Incorporation Assay, Two Tailed Test

CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *
Figure Legend Snippet: CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *

Techniques Used: Irradiation, Mouse Assay, Two Tailed Test

CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Expressing, FACS, Staining, Mouse Assay, Binding Assay, Chromatin Immunoprecipitation, Cell Culture, Two Tailed Test

34) Product Images from "CD74 is a regulator of hematopoietic stem cell maintenance"

Article Title: CD74 is a regulator of hematopoietic stem cell maintenance

Journal: PLoS Biology

doi: 10.1371/journal.pbio.3001121

CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, Two Tailed Test

CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, Transplantation Assay, Two Tailed Test

Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Mouse Assay, Derivative Assay, Purification, Expressing, FACS, Two Tailed Test

CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, FACS, Staining, Two Tailed Test

CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *
Figure Legend Snippet: CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *

Techniques Used: Cell Culture, Incubation, Blocking Assay, FACS, Injection, Mouse Assay

Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p
Figure Legend Snippet: Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p

Techniques Used: FACS, Expressing, Mouse Assay, Injection, Staining, BrdU Incorporation Assay, Two Tailed Test

CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *
Figure Legend Snippet: CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *

Techniques Used: Irradiation, Mouse Assay, Two Tailed Test

CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Expressing, FACS, Staining, Mouse Assay, Binding Assay, Chromatin Immunoprecipitation, Cell Culture, Two Tailed Test

35) Product Images from "CD74 is a regulator of hematopoietic stem cell maintenance"

Article Title: CD74 is a regulator of hematopoietic stem cell maintenance

Journal: PLoS Biology

doi: 10.1371/journal.pbio.3001121

CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, Two Tailed Test

CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, Transplantation Assay, Two Tailed Test

Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Mouse Assay, Derivative Assay, Purification, Expressing, FACS, Two Tailed Test

CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, FACS, Staining, Two Tailed Test

CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *
Figure Legend Snippet: CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *

Techniques Used: Cell Culture, Incubation, Blocking Assay, FACS, Injection, Mouse Assay

Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p
Figure Legend Snippet: Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p

Techniques Used: FACS, Expressing, Mouse Assay, Injection, Staining, BrdU Incorporation Assay, Two Tailed Test

CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *
Figure Legend Snippet: CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *

Techniques Used: Irradiation, Mouse Assay, Two Tailed Test

CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Expressing, FACS, Staining, Mouse Assay, Binding Assay, Chromatin Immunoprecipitation, Cell Culture, Two Tailed Test

36) Product Images from "Milatuzumab-Conjugated Liposomes as Targeted Dexamethasone Carriers for Therapeutic Delivery in CD74+ B-cell Malignancies"

Article Title: Milatuzumab-Conjugated Liposomes as Targeted Dexamethasone Carriers for Therapeutic Delivery in CD74+ B-cell Malignancies

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

doi: 10.1158/1078-0432.CCR-12-2046

Localization of CD74-ILs in target cells. CD74-ILs, after 1-hour incubation with Raji cells (A, B, G, J) visualized by confocal microscopy. CD74-ILs are observed inside the cells and also on the cell membrane. Controls such as IgG-ILs (C and L) and nontargeted liposomes (D, E, K) did not enter/bind the cells. CD74-ILs in CD74 − cell line Jurkat (I) did not bind/enter the cells. All lipids are R18-labeled.
Figure Legend Snippet: Localization of CD74-ILs in target cells. CD74-ILs, after 1-hour incubation with Raji cells (A, B, G, J) visualized by confocal microscopy. CD74-ILs are observed inside the cells and also on the cell membrane. Controls such as IgG-ILs (C and L) and nontargeted liposomes (D, E, K) did not enter/bind the cells. CD74-ILs in CD74 − cell line Jurkat (I) did not bind/enter the cells. All lipids are R18-labeled.

Techniques Used: Incubation, Confocal Microscopy, Labeling

CD74-IL-DEX decreases viability and mitochondrial activity in CLL cells. Primary CLL cells ( n = 14) were treated for 24 hours with dexamethasone (10 μmol/L) in free or liposomal forms with cross-linked milatuzumab (CD74+Fc) and all CD74 concentrations at 5 μg/mL (A). Flow cytometric analysis of percentage of PI-positive cells indicated that CD74-IL-DEX kills significantly more B-CLL cells than CD74-ILs ( P
Figure Legend Snippet: CD74-IL-DEX decreases viability and mitochondrial activity in CLL cells. Primary CLL cells ( n = 14) were treated for 24 hours with dexamethasone (10 μmol/L) in free or liposomal forms with cross-linked milatuzumab (CD74+Fc) and all CD74 concentrations at 5 μg/mL (A). Flow cytometric analysis of percentage of PI-positive cells indicated that CD74-IL-DEX kills significantly more B-CLL cells than CD74-ILs ( P

Techniques Used: Activity Assay, Flow Cytometry

CD74-IL-DEX and L-DEX increase cellular GilZ level. Immunoblot analysis of GilZ induction in whole-cell lysates isolated from Raji cells treated with different formulations of DEX at 10 μmol/L and equivalent CD74.
Figure Legend Snippet: CD74-IL-DEX and L-DEX increase cellular GilZ level. Immunoblot analysis of GilZ induction in whole-cell lysates isolated from Raji cells treated with different formulations of DEX at 10 μmol/L and equivalent CD74.

Techniques Used: Isolation

CD74-ILs bind to and are internalized into CD74 + Raji cells. CD74-ILs labeled with calcein are shown by flow cytometry to bind to CD74( + ) Raji B cells (A) but not CD74 − Jurkat T cells (B). Nonspecific IgG-ILs do not bind to Raji cells (AandB). Internalization of CD74-ILs and anti-CD74 ( n = 3) is shown in Raji cells (C), over time portrayed by change in MFI. The results were normalized to IgG isotype and IgG-ILs.
Figure Legend Snippet: CD74-ILs bind to and are internalized into CD74 + Raji cells. CD74-ILs labeled with calcein are shown by flow cytometry to bind to CD74( + ) Raji B cells (A) but not CD74 − Jurkat T cells (B). Nonspecific IgG-ILs do not bind to Raji cells (AandB). Internalization of CD74-ILs and anti-CD74 ( n = 3) is shown in Raji cells (C), over time portrayed by change in MFI. The results were normalized to IgG isotype and IgG-ILs.

Techniques Used: Labeling, Flow Cytometry, Cytometry

37) Product Images from "CD74 is a regulator of hematopoietic stem cell maintenance"

Article Title: CD74 is a regulator of hematopoietic stem cell maintenance

Journal: PLoS Biology

doi: 10.1371/journal.pbio.3001121

CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, Two Tailed Test

CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, Transplantation Assay, Two Tailed Test

Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Mouse Assay, Derivative Assay, Purification, Expressing, FACS, Two Tailed Test

CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, FACS, Staining, Two Tailed Test

CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *
Figure Legend Snippet: CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *

Techniques Used: Cell Culture, Incubation, Blocking Assay, FACS, Injection, Mouse Assay

Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p
Figure Legend Snippet: Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p

Techniques Used: FACS, Expressing, Mouse Assay, Injection, Staining, BrdU Incorporation Assay, Two Tailed Test

CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *
Figure Legend Snippet: CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *

Techniques Used: Irradiation, Mouse Assay, Two Tailed Test

CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Expressing, FACS, Staining, Mouse Assay, Binding Assay, Chromatin Immunoprecipitation, Cell Culture, Two Tailed Test

38) Product Images from "CD74 is a regulator of hematopoietic stem cell maintenance"

Article Title: CD74 is a regulator of hematopoietic stem cell maintenance

Journal: PLoS Biology

doi: 10.1371/journal.pbio.3001121

CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, Two Tailed Test

CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, Transplantation Assay, Two Tailed Test

Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Mouse Assay, Derivative Assay, Purification, Expressing, FACS, Two Tailed Test

CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, FACS, Staining, Two Tailed Test

CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *
Figure Legend Snippet: CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *

Techniques Used: Cell Culture, Incubation, Blocking Assay, FACS, Injection, Mouse Assay

Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p
Figure Legend Snippet: Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p

Techniques Used: FACS, Expressing, Mouse Assay, Injection, Staining, BrdU Incorporation Assay, Two Tailed Test

CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *
Figure Legend Snippet: CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *

Techniques Used: Irradiation, Mouse Assay, Two Tailed Test

CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Expressing, FACS, Staining, Mouse Assay, Binding Assay, Chromatin Immunoprecipitation, Cell Culture, Two Tailed Test

39) Product Images from "CD74 is a regulator of hematopoietic stem cell maintenance"

Article Title: CD74 is a regulator of hematopoietic stem cell maintenance

Journal: PLoS Biology

doi: 10.1371/journal.pbio.3001121

CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, Two Tailed Test

CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, Transplantation Assay, Two Tailed Test

Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Mouse Assay, Derivative Assay, Purification, Expressing, FACS, Two Tailed Test

CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, FACS, Staining, Two Tailed Test

CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *
Figure Legend Snippet: CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *

Techniques Used: Cell Culture, Incubation, Blocking Assay, FACS, Injection, Mouse Assay

Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p
Figure Legend Snippet: Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p

Techniques Used: FACS, Expressing, Mouse Assay, Injection, Staining, BrdU Incorporation Assay, Two Tailed Test

CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *
Figure Legend Snippet: CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *

Techniques Used: Irradiation, Mouse Assay, Two Tailed Test

CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Expressing, FACS, Staining, Mouse Assay, Binding Assay, Chromatin Immunoprecipitation, Cell Culture, Two Tailed Test

40) Product Images from "CD74 is a regulator of hematopoietic stem cell maintenance"

Article Title: CD74 is a regulator of hematopoietic stem cell maintenance

Journal: PLoS Biology

doi: 10.1371/journal.pbio.3001121

CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, Two Tailed Test

CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, Transplantation Assay, Two Tailed Test

Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Mouse Assay, Derivative Assay, Purification, Expressing, FACS, Two Tailed Test

CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Irradiation, Mouse Assay, Derivative Assay, FACS, Staining, Two Tailed Test

CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *
Figure Legend Snippet: CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *

Techniques Used: Cell Culture, Incubation, Blocking Assay, FACS, Injection, Mouse Assay

Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p
Figure Legend Snippet: Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p

Techniques Used: FACS, Expressing, Mouse Assay, Injection, Staining, BrdU Incorporation Assay, Two Tailed Test

CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *
Figure Legend Snippet: CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *

Techniques Used: Irradiation, Mouse Assay, Two Tailed Test

CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p
Figure Legend Snippet: CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p

Techniques Used: Expressing, FACS, Staining, Mouse Assay, Binding Assay, Chromatin Immunoprecipitation, Cell Culture, Two Tailed Test

Related Articles

Expressing:

Article Title: CD74 is a regulator of hematopoietic stem cell maintenance
Article Snippet: .. MIF/CD74 axis regulates CD18 expression. (A) FACS analysis of LSK from WT and CD74−/− mice for CD18; n = 6, Data A in S13. (B) FACS analysis of LSK from WT and MIF−/− mice for CD18; n = 3, Data B in . (C) WT and CD74−/− BM were cultured with MIF inhibitor (ISO-1) for 48 h, and percent CD18 on LSK was analyzed; n = 6, Data C in (D) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF−/− (CD45.2) total BM for 48 h, the percent CD18 on LSK cells (CD45.1) was analyzed by FACS; n = 8, Data D in (E) LSK and CD34-/LSK populations from WT and CD74−/− mice were analyzed for cell surface expression of CD11A, CD11B, and CD11C; n = 6 by FACS. .. Data E in . (F) WT and CD74−/− BM were cultured with the MIF inhibitor (ISO-1) for 48 h, and percent CD11B on LSK and CD34-/LSK was analyzed by FACS; n = 4, Data F in .

Article Title: CD74 is a regulator of hematopoietic stem cell maintenance
Article Snippet: Supporting information (A–D) Gating strategy of HSPCs. (E) Sorted CD34-/LSK cells were analyzed for CD74 mRNA levels in WT and CD74−/− mice; n = 3. .. Bars show the DESeq2 normalized counts for the CD74 gene, Data A in . (F) Lin- populations from WT and CD74−/− mice were analyzed for cKit and Sca-1 expression by FACS; n = 15, Data B in . (G) C57BL/6 and CD74−/− mice were crossed to obtain WT and CD74−/− littermate mice. .. Percent of CD34-/LSK from WT and CD74−/− littermates; n = 3, Data C in . (H) Sorted CD34-/LSK cells were analyzed for MIF mRNA levels in WT and CD74−/− mice; n = 3.

FACS:

Article Title: CD74 is a regulator of hematopoietic stem cell maintenance
Article Snippet: .. MIF/CD74 axis regulates CD18 expression. (A) FACS analysis of LSK from WT and CD74−/− mice for CD18; n = 6, Data A in S13. (B) FACS analysis of LSK from WT and MIF−/− mice for CD18; n = 3, Data B in . (C) WT and CD74−/− BM were cultured with MIF inhibitor (ISO-1) for 48 h, and percent CD18 on LSK was analyzed; n = 6, Data C in (D) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF−/− (CD45.2) total BM for 48 h, the percent CD18 on LSK cells (CD45.1) was analyzed by FACS; n = 8, Data D in (E) LSK and CD34-/LSK populations from WT and CD74−/− mice were analyzed for cell surface expression of CD11A, CD11B, and CD11C; n = 6 by FACS. .. Data E in . (F) WT and CD74−/− BM were cultured with the MIF inhibitor (ISO-1) for 48 h, and percent CD11B on LSK and CD34-/LSK was analyzed by FACS; n = 4, Data F in .

Article Title: CD74 is a regulator of hematopoietic stem cell maintenance
Article Snippet: Supporting information (A–D) Gating strategy of HSPCs. (E) Sorted CD34-/LSK cells were analyzed for CD74 mRNA levels in WT and CD74−/− mice; n = 3. .. Bars show the DESeq2 normalized counts for the CD74 gene, Data A in . (F) Lin- populations from WT and CD74−/− mice were analyzed for cKit and Sca-1 expression by FACS; n = 15, Data B in . (G) C57BL/6 and CD74−/− mice were crossed to obtain WT and CD74−/− littermate mice. .. Percent of CD34-/LSK from WT and CD74−/− littermates; n = 3, Data C in . (H) Sorted CD34-/LSK cells were analyzed for MIF mRNA levels in WT and CD74−/− mice; n = 3.

Article Title: CD74 is a regulator of hematopoietic stem cell maintenance
Article Snippet: .. Library construction and sequencing HSCs (CD34-/LSK) (1*103 cells) were sorted from WT and CD74−/− mice (BD FACS Aria III). .. Sequencing libraries were prepared using the SMART-Seq v4 Ultra Low Input RNA kit (Clonetech, United States of America).

Mouse Assay:

Article Title: CD74 is a regulator of hematopoietic stem cell maintenance
Article Snippet: .. MIF/CD74 axis regulates CD18 expression. (A) FACS analysis of LSK from WT and CD74−/− mice for CD18; n = 6, Data A in S13. (B) FACS analysis of LSK from WT and MIF−/− mice for CD18; n = 3, Data B in . (C) WT and CD74−/− BM were cultured with MIF inhibitor (ISO-1) for 48 h, and percent CD18 on LSK was analyzed; n = 6, Data C in (D) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF−/− (CD45.2) total BM for 48 h, the percent CD18 on LSK cells (CD45.1) was analyzed by FACS; n = 8, Data D in (E) LSK and CD34-/LSK populations from WT and CD74−/− mice were analyzed for cell surface expression of CD11A, CD11B, and CD11C; n = 6 by FACS. .. Data E in . (F) WT and CD74−/− BM were cultured with the MIF inhibitor (ISO-1) for 48 h, and percent CD11B on LSK and CD34-/LSK was analyzed by FACS; n = 4, Data F in .

Article Title: CD74 is a regulator of hematopoietic stem cell maintenance
Article Snippet: Supporting information (A–D) Gating strategy of HSPCs. (E) Sorted CD34-/LSK cells were analyzed for CD74 mRNA levels in WT and CD74−/− mice; n = 3. .. Bars show the DESeq2 normalized counts for the CD74 gene, Data A in . (F) Lin- populations from WT and CD74−/− mice were analyzed for cKit and Sca-1 expression by FACS; n = 15, Data B in . (G) C57BL/6 and CD74−/− mice were crossed to obtain WT and CD74−/− littermate mice. .. Percent of CD34-/LSK from WT and CD74−/− littermates; n = 3, Data C in . (H) Sorted CD34-/LSK cells were analyzed for MIF mRNA levels in WT and CD74−/− mice; n = 3.

Article Title: CD74 is a regulator of hematopoietic stem cell maintenance
Article Snippet: .. Short-term chimera: Lethally irradiated (950 Rad) CD45.1 recipient mice were reconstituted with 25*106 of either WT(CD45.2) or CD74−/−(CD45.2) BM cells. ..

Article Title: CD74 is a regulator of hematopoietic stem cell maintenance
Article Snippet: Sorting of the LSK CD45.1 WT and CD45.2 CD74−/− cells was performed using a FACS Aria II system (BD Bioscience, USA), following enrichment using CD117 (c-Kit) MicroBeads (cat: 130-091-224) on LS MACS Separation Columns (cat: 130-042-401), both obtained from Miltenyi Biotec, UK. .. Generation of chimeric mice For the microenvironment experiment: Lethally irradiated (950 Rad) C57BL/6 (WT) recipient mice were reconstituted with 5*106 WT or CD74−/− BM cells. .. Additionally, lethally irradiated (950 rad) CD74−/− recipient mice on a C57BL/6 background were reconstituted with 5*106 WT or CD74−/− total BM cells.

Article Title: CD74 is a regulator of hematopoietic stem cell maintenance
Article Snippet: .. Library construction and sequencing HSCs (CD34-/LSK) (1*103 cells) were sorted from WT and CD74−/− mice (BD FACS Aria III). .. Sequencing libraries were prepared using the SMART-Seq v4 Ultra Low Input RNA kit (Clonetech, United States of America).

Cell Culture:

Article Title: CD74 is a regulator of hematopoietic stem cell maintenance
Article Snippet: .. MIF/CD74 axis regulates CD18 expression. (A) FACS analysis of LSK from WT and CD74−/− mice for CD18; n = 6, Data A in S13. (B) FACS analysis of LSK from WT and MIF−/− mice for CD18; n = 3, Data B in . (C) WT and CD74−/− BM were cultured with MIF inhibitor (ISO-1) for 48 h, and percent CD18 on LSK was analyzed; n = 6, Data C in (D) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF−/− (CD45.2) total BM for 48 h, the percent CD18 on LSK cells (CD45.1) was analyzed by FACS; n = 8, Data D in (E) LSK and CD34-/LSK populations from WT and CD74−/− mice were analyzed for cell surface expression of CD11A, CD11B, and CD11C; n = 6 by FACS. .. Data E in . (F) WT and CD74−/− BM were cultured with the MIF inhibitor (ISO-1) for 48 h, and percent CD11B on LSK and CD34-/LSK was analyzed by FACS; n = 4, Data F in .

other:

Article Title: CD74 is a regulator of hematopoietic stem cell maintenance
Article Snippet: 1) The authors convincingly demonstrate that the CD74 deletion in non-hematopoietic cells does not contribute to the phenotype, but the experiments do not exclude the possibility that CD74 in other hematopoietic cells plays a role.

Article Title: CD74 is a regulator of hematopoietic stem cell maintenance
Article Snippet: 6) In their previous paper (Gore Y et al, J Biol Chem, 2008), it shows that MIF binds to a complex of CD74 and CD44, resulting in initiation of a signaling pathway.

Article Title: CD74 is a regulator of hematopoietic stem cell maintenance
Article Snippet: 2) According to the result of the effect of CD74 blockade and the role of CD74 in haematological recovery from chemotherapy with 5-FU treatment, it helps improve clinical insight into bone marrow transplant protocols and enable improved engraftment.

Irradiation:

Article Title: CD74 is a regulator of hematopoietic stem cell maintenance
Article Snippet: .. Short-term chimera: Lethally irradiated (950 Rad) CD45.1 recipient mice were reconstituted with 25*106 of either WT(CD45.2) or CD74−/−(CD45.2) BM cells. ..

Article Title: CD74 is a regulator of hematopoietic stem cell maintenance
Article Snippet: Sorting of the LSK CD45.1 WT and CD45.2 CD74−/− cells was performed using a FACS Aria II system (BD Bioscience, USA), following enrichment using CD117 (c-Kit) MicroBeads (cat: 130-091-224) on LS MACS Separation Columns (cat: 130-042-401), both obtained from Miltenyi Biotec, UK. .. Generation of chimeric mice For the microenvironment experiment: Lethally irradiated (950 Rad) C57BL/6 (WT) recipient mice were reconstituted with 5*106 WT or CD74−/− BM cells. .. Additionally, lethally irradiated (950 rad) CD74−/− recipient mice on a C57BL/6 background were reconstituted with 5*106 WT or CD74−/− total BM cells.

Sequencing:

Article Title: CD74 is a regulator of hematopoietic stem cell maintenance
Article Snippet: .. Library construction and sequencing HSCs (CD34-/LSK) (1*103 cells) were sorted from WT and CD74−/− mice (BD FACS Aria III). .. Sequencing libraries were prepared using the SMART-Seq v4 Ultra Low Input RNA kit (Clonetech, United States of America).

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    Becton Dickinson bd facs aria ii
    Fluorescence analysis of 2-NBDG uptake by flow cytometry. <t>FACS</t> analysis of 2-NBDG uptake in differentiated L6 cells by plotting cell count against <t>FITC</t> revealed that 8%, 8.1% and 30% of cells uptake 2-NBDG in control, TBHP and Rosiglitazone treated cells respectively whereas 30.6%, 33.1%, 28%, 32% of cells uptake 2-NBDG, pretreated with two different concentrations (10 and 100 μM) of Naringin along with/without TBHP respectively. Each value represents mean ± SD (standard deviation) from triplicate measurements (n = 3) of three different experiments. Significance test between various groups was determined by using one way ANOVA followed by Duncan’s multiple range test. * P≤0.05 versus control.
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    Becton Dickinson anti mhc class ii
    Turnover of SI-LP and MLN <t>CD103</t> + and CD103 − DC. (A) Mice were injected i.p. with 2 mg BrdU, and the percentage of BrdU + CD103 + and CD103 − DCs <t>(MHC</t> class II + CD11c + ) in the SI-LP and MLN was determined by flow cytometry at the times indicated. Results are the mean and SD of three to seven independent experiments with two mice in each time point except the 72-h time point, which was performed once. (B) BrdU and Ki67 staining on SI-LP and MLN CD103 + and CD103 − DCs (MHC class II + CD11c + ) was assessed by flow cytometry 3 and 24 h after BrdU injection. Plots are from one representative experiment of three performed. (C) SI from CD45.2 + mice (graft) was transplanted into CD45.1 + recipients (host) as previously described ( 32 ). At days 6 and 45, host and graft intestine were sectioned and stained with antibodies to CD45.2 (red), CD11c (blue), and MHC class II (green). Immunohistochemistry of the 45-d graft is shown. Arrows point to host-derived CD45.2 − MHC class II hi CD11c + DCs. Bar, 25 μm. The graph is percentage of host-derived (CD45.2 negative) CD11c + MHC class II hi cells in graft and host small intestinal villus. Results are mean and SD ( n = 3 mice per group).
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    Becton Dickinson facs canto ii flow cytometer
    Flow <t>cytometry</t> pulse width measurements correlate well with cell diameter. Fluorescence activated cell sorting <t>(FACS)</t> of C. vulgaris cells was used to sort a mixture of cells and beads, according to SSC-W range. Cells and polystyrene marker beads were photographed after sorting (a) in the range SSC-W = 60000–65000 (cells grown in TP), and (b) in the range SSC-W = 70000–80000 (cells grown in TP+100 mM glucose). Representative beads (black arrows) and cells (grey arrows) marked are indicated. (c) Diameters were measured microscopically using IMARIS software for ∼100 cells or beads after sorting and the mean, standard deviation and range was plotted for each population of cells and the marker beads.
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    Becton Dickinson bd facsaria ii
    Adjuvanticity of FlgE to Soluble Antigen in Mice. ( A–C ) Naïve OVA-specific T cells were purified from the spleens and inguinal lymph nodes of CD45.1 × OTII F1 mice by sorting using various antibodies to surface markers, labeled with eFluor450 and transferred into female C57BL/6 mice at 1 × 10 6 cells/mouse via tail vein injection. Twenty-four hours later, the recipients were treated or immunized via subcutaneous injection at the base of the tail with one of eight compositions, namely 1 μg OVA, 50 μg CpG-1826, 50 μg FlgE, 50 μg FlgEM each alone, or OVA plus the three stimulants individually. Control groups received 100 μL PBS. Three days later, the mice were sacrificed, and the draining inguinal lymph nodes were isolated and photographed ( A ). Single-cell suspension were made for staining with CD45.1 plus CD4 and then run on a BD <t>FACSAria</t> II and analyzed for eFluor450 intensity to quantify the proliferation of OVA-specific T cells ( B,C ). ( D ) To measure the humoral response, WT C57BL/6 mice were immunized with above 8 compositions except for increasing OVA doses to 100 μg. Two weeks later, the mice were sacrificed for serum harvest, and anti-OVA titers were measured using an ELISA as described in Materials and Methods. Data are representatives of two independent experiments that showed similar results. n = 3 mice for panel A–C, and n = 4 mice for panel D. *P
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    Fluorescence analysis of 2-NBDG uptake by flow cytometry. FACS analysis of 2-NBDG uptake in differentiated L6 cells by plotting cell count against FITC revealed that 8%, 8.1% and 30% of cells uptake 2-NBDG in control, TBHP and Rosiglitazone treated cells respectively whereas 30.6%, 33.1%, 28%, 32% of cells uptake 2-NBDG, pretreated with two different concentrations (10 and 100 μM) of Naringin along with/without TBHP respectively. Each value represents mean ± SD (standard deviation) from triplicate measurements (n = 3) of three different experiments. Significance test between various groups was determined by using one way ANOVA followed by Duncan’s multiple range test. * P≤0.05 versus control.

    Journal: PLoS ONE

    Article Title: Preconditioning L6 Muscle Cells with Naringin Ameliorates Oxidative Stress and Increases Glucose Uptake

    doi: 10.1371/journal.pone.0132429

    Figure Lengend Snippet: Fluorescence analysis of 2-NBDG uptake by flow cytometry. FACS analysis of 2-NBDG uptake in differentiated L6 cells by plotting cell count against FITC revealed that 8%, 8.1% and 30% of cells uptake 2-NBDG in control, TBHP and Rosiglitazone treated cells respectively whereas 30.6%, 33.1%, 28%, 32% of cells uptake 2-NBDG, pretreated with two different concentrations (10 and 100 μM) of Naringin along with/without TBHP respectively. Each value represents mean ± SD (standard deviation) from triplicate measurements (n = 3) of three different experiments. Significance test between various groups was determined by using one way ANOVA followed by Duncan’s multiple range test. * P≤0.05 versus control.

    Article Snippet: Samples were analyzed using BD FACS Aria II (BD Biosciences) at FITC range (excitation 490 nm, emission 525 nm band pass filter).

    Techniques: Fluorescence, Flow Cytometry, Cytometry, FACS, Cell Counting, Standard Deviation

    Turnover of SI-LP and MLN CD103 + and CD103 − DC. (A) Mice were injected i.p. with 2 mg BrdU, and the percentage of BrdU + CD103 + and CD103 − DCs (MHC class II + CD11c + ) in the SI-LP and MLN was determined by flow cytometry at the times indicated. Results are the mean and SD of three to seven independent experiments with two mice in each time point except the 72-h time point, which was performed once. (B) BrdU and Ki67 staining on SI-LP and MLN CD103 + and CD103 − DCs (MHC class II + CD11c + ) was assessed by flow cytometry 3 and 24 h after BrdU injection. Plots are from one representative experiment of three performed. (C) SI from CD45.2 + mice (graft) was transplanted into CD45.1 + recipients (host) as previously described ( 32 ). At days 6 and 45, host and graft intestine were sectioned and stained with antibodies to CD45.2 (red), CD11c (blue), and MHC class II (green). Immunohistochemistry of the 45-d graft is shown. Arrows point to host-derived CD45.2 − MHC class II hi CD11c + DCs. Bar, 25 μm. The graph is percentage of host-derived (CD45.2 negative) CD11c + MHC class II hi cells in graft and host small intestinal villus. Results are mean and SD ( n = 3 mice per group).

    Journal: The Journal of Experimental Medicine

    Article Title: Small intestinal CD103+ dendritic cells display unique functional properties that are conserved between mice and humans

    doi: 10.1084/jem.20080414

    Figure Lengend Snippet: Turnover of SI-LP and MLN CD103 + and CD103 − DC. (A) Mice were injected i.p. with 2 mg BrdU, and the percentage of BrdU + CD103 + and CD103 − DCs (MHC class II + CD11c + ) in the SI-LP and MLN was determined by flow cytometry at the times indicated. Results are the mean and SD of three to seven independent experiments with two mice in each time point except the 72-h time point, which was performed once. (B) BrdU and Ki67 staining on SI-LP and MLN CD103 + and CD103 − DCs (MHC class II + CD11c + ) was assessed by flow cytometry 3 and 24 h after BrdU injection. Plots are from one representative experiment of three performed. (C) SI from CD45.2 + mice (graft) was transplanted into CD45.1 + recipients (host) as previously described ( 32 ). At days 6 and 45, host and graft intestine were sectioned and stained with antibodies to CD45.2 (red), CD11c (blue), and MHC class II (green). Immunohistochemistry of the 45-d graft is shown. Arrows point to host-derived CD45.2 − MHC class II hi CD11c + DCs. Bar, 25 μm. The graph is percentage of host-derived (CD45.2 negative) CD11c + MHC class II hi cells in graft and host small intestinal villus. Results are mean and SD ( n = 3 mice per group).

    Article Snippet: Cell staining reagents The following reagents were used to stain murine cells: unconjugated or directly conjugated anti-CD103 (M290), anti-α4β7 (DATK32), anti-CD62L (MEL-14), anti-MHC class II (2G9), anti-CD11c (N418), and rat IgG2a and IgG2b isotype controls (BD Biosciences); perCP-Cy5.5, Alexa 700–conjugated anti-CD11c (N418), and Pacific blue–conjugated anti–MHC Class II (M5/114.15.2; BioLegend); anti-CCR7 (4B12; eBiosciences); recombinant mouse E-selectin human Fc chimera (R & D Systems); anti-FcRII/III (2.4G2), anti-CD40 (FGK45), anti-CD86 (GL1), and anti-CCR9 antibody (7E7, ( ); purified from hybridomas); Cy5-labeled donkey anti–human IgG F(ab′)2 (Jackson ImmunoResearch Laboratories); biotinylated mouse anti–rat IgG2a (RG7/1.30), biotinylated mouse anti–rat IgG2b (G15-337), PE-labeled Ki67, streptavidin-conjugated PE, PECy7, PerCP Cy5.5, and the BrdU FITC flow kit (BD Biosciences); streptavidin–alexa 488 (Invitrogen); and DAPI (Sigma-Aldrich).

    Techniques: Mouse Assay, Injection, Flow Cytometry, Cytometry, Staining, Immunohistochemistry, Derivative Assay

    Localization of CD103 + and CD103 − MHC class II + CD11c + cells within the small intestinal mucosa. (A and B) Expression of CD103 by DCs in small intestinal villus and SILT. (A) Images show overlays of CD103 (red) in combination with CD11c (blue) and MHC class II (green), with insets showing DAPI staining. Arrows point toward MHC class II hi CD11c + DCs coexpressing CD103. Bars, 50 μm. (B) The graph is percentage of MHC class II hi CD11c + cells in SI villus or dome region of SILT that express CD103. Results are mean and SD ( n = 5 mice per group). ***, P

    Journal: The Journal of Experimental Medicine

    Article Title: Small intestinal CD103+ dendritic cells display unique functional properties that are conserved between mice and humans

    doi: 10.1084/jem.20080414

    Figure Lengend Snippet: Localization of CD103 + and CD103 − MHC class II + CD11c + cells within the small intestinal mucosa. (A and B) Expression of CD103 by DCs in small intestinal villus and SILT. (A) Images show overlays of CD103 (red) in combination with CD11c (blue) and MHC class II (green), with insets showing DAPI staining. Arrows point toward MHC class II hi CD11c + DCs coexpressing CD103. Bars, 50 μm. (B) The graph is percentage of MHC class II hi CD11c + cells in SI villus or dome region of SILT that express CD103. Results are mean and SD ( n = 5 mice per group). ***, P

    Article Snippet: Cell staining reagents The following reagents were used to stain murine cells: unconjugated or directly conjugated anti-CD103 (M290), anti-α4β7 (DATK32), anti-CD62L (MEL-14), anti-MHC class II (2G9), anti-CD11c (N418), and rat IgG2a and IgG2b isotype controls (BD Biosciences); perCP-Cy5.5, Alexa 700–conjugated anti-CD11c (N418), and Pacific blue–conjugated anti–MHC Class II (M5/114.15.2; BioLegend); anti-CCR7 (4B12; eBiosciences); recombinant mouse E-selectin human Fc chimera (R & D Systems); anti-FcRII/III (2.4G2), anti-CD40 (FGK45), anti-CD86 (GL1), and anti-CCR9 antibody (7E7, ( ); purified from hybridomas); Cy5-labeled donkey anti–human IgG F(ab′)2 (Jackson ImmunoResearch Laboratories); biotinylated mouse anti–rat IgG2a (RG7/1.30), biotinylated mouse anti–rat IgG2b (G15-337), PE-labeled Ki67, streptavidin-conjugated PE, PECy7, PerCP Cy5.5, and the BrdU FITC flow kit (BD Biosciences); streptavidin–alexa 488 (Invitrogen); and DAPI (Sigma-Aldrich).

    Techniques: Expressing, Staining, Mouse Assay

    Flow cytometry pulse width measurements correlate well with cell diameter. Fluorescence activated cell sorting (FACS) of C. vulgaris cells was used to sort a mixture of cells and beads, according to SSC-W range. Cells and polystyrene marker beads were photographed after sorting (a) in the range SSC-W = 60000–65000 (cells grown in TP), and (b) in the range SSC-W = 70000–80000 (cells grown in TP+100 mM glucose). Representative beads (black arrows) and cells (grey arrows) marked are indicated. (c) Diameters were measured microscopically using IMARIS software for ∼100 cells or beads after sorting and the mean, standard deviation and range was plotted for each population of cells and the marker beads.

    Journal: PLoS ONE

    Article Title: Flow Cytometry Pulse Width Data Enables Rapid and Sensitive Estimation of Biomass Dry Weight in the Microalgae Chlamydomonas reinhardtii and Chlorella vulgaris

    doi: 10.1371/journal.pone.0097269

    Figure Lengend Snippet: Flow cytometry pulse width measurements correlate well with cell diameter. Fluorescence activated cell sorting (FACS) of C. vulgaris cells was used to sort a mixture of cells and beads, according to SSC-W range. Cells and polystyrene marker beads were photographed after sorting (a) in the range SSC-W = 60000–65000 (cells grown in TP), and (b) in the range SSC-W = 70000–80000 (cells grown in TP+100 mM glucose). Representative beads (black arrows) and cells (grey arrows) marked are indicated. (c) Diameters were measured microscopically using IMARIS software for ∼100 cells or beads after sorting and the mean, standard deviation and range was plotted for each population of cells and the marker beads.

    Article Snippet: Flow cytometry Cytometric analysis was carried out using a BD FACS Canto II Flow Cytometer (BD, San Jose, USA) fitted with a high throughput system (HTS) to analyse multiwell plate samples.

    Techniques: Flow Cytometry, Cytometry, Fluorescence, FACS, Marker, Software, Standard Deviation

    Adjuvanticity of FlgE to Soluble Antigen in Mice. ( A–C ) Naïve OVA-specific T cells were purified from the spleens and inguinal lymph nodes of CD45.1 × OTII F1 mice by sorting using various antibodies to surface markers, labeled with eFluor450 and transferred into female C57BL/6 mice at 1 × 10 6 cells/mouse via tail vein injection. Twenty-four hours later, the recipients were treated or immunized via subcutaneous injection at the base of the tail with one of eight compositions, namely 1 μg OVA, 50 μg CpG-1826, 50 μg FlgE, 50 μg FlgEM each alone, or OVA plus the three stimulants individually. Control groups received 100 μL PBS. Three days later, the mice were sacrificed, and the draining inguinal lymph nodes were isolated and photographed ( A ). Single-cell suspension were made for staining with CD45.1 plus CD4 and then run on a BD FACSAria II and analyzed for eFluor450 intensity to quantify the proliferation of OVA-specific T cells ( B,C ). ( D ) To measure the humoral response, WT C57BL/6 mice were immunized with above 8 compositions except for increasing OVA doses to 100 μg. Two weeks later, the mice were sacrificed for serum harvest, and anti-OVA titers were measured using an ELISA as described in Materials and Methods. Data are representatives of two independent experiments that showed similar results. n = 3 mice for panel A–C, and n = 4 mice for panel D. *P

    Journal: Scientific Reports

    Article Title: Flagellar Hooks and Hook Protein FlgE Participate in Host Microbe Interactions at Immunological Level

    doi: 10.1038/s41598-017-01619-1

    Figure Lengend Snippet: Adjuvanticity of FlgE to Soluble Antigen in Mice. ( A–C ) Naïve OVA-specific T cells were purified from the spleens and inguinal lymph nodes of CD45.1 × OTII F1 mice by sorting using various antibodies to surface markers, labeled with eFluor450 and transferred into female C57BL/6 mice at 1 × 10 6 cells/mouse via tail vein injection. Twenty-four hours later, the recipients were treated or immunized via subcutaneous injection at the base of the tail with one of eight compositions, namely 1 μg OVA, 50 μg CpG-1826, 50 μg FlgE, 50 μg FlgEM each alone, or OVA plus the three stimulants individually. Control groups received 100 μL PBS. Three days later, the mice were sacrificed, and the draining inguinal lymph nodes were isolated and photographed ( A ). Single-cell suspension were made for staining with CD45.1 plus CD4 and then run on a BD FACSAria II and analyzed for eFluor450 intensity to quantify the proliferation of OVA-specific T cells ( B,C ). ( D ) To measure the humoral response, WT C57BL/6 mice were immunized with above 8 compositions except for increasing OVA doses to 100 μg. Two weeks later, the mice were sacrificed for serum harvest, and anti-OVA titers were measured using an ELISA as described in Materials and Methods. Data are representatives of two independent experiments that showed similar results. n = 3 mice for panel A–C, and n = 4 mice for panel D. *P

    Article Snippet: Cells were run on a BD FACSAria II and analyzed for eFluor450 intensity to reflect proliferation.

    Techniques: Mouse Assay, Purification, Labeling, Injection, Isolation, Staining, Enzyme-linked Immunosorbent Assay