Journal: The Journal of Biological Chemistry

Article Title: A STUB1 ubiquitin ligase/CHIC2 protein complex negatively regulates the IL-3, IL-5, and GM-CSF cytokine receptor common β chain (CSF2RB) protein stability

doi: 10.1016/j.jbc.2022.102484

Figure Lengend Snippet: Ubiquitin ligase–specific and whole-genome CRISPR-Cas9 screens identify STUB1 and CHIC2 as regulators of CSF2RB protein stability in TF1 and 32D cell lines. A , diagram of CSF2RB reporter. B , bar graph showing normalized GFP/mCherry ratio of CSF2RB reporter in TF1 Cas9 cells treated with DMSO control, 1 μM MLN7243 (E1 inhibitor), 5 μM MLN4924 (neddylation inhibitor), or 10 μM MG132 (proteasomal inhibitor) for 4 h in 5 ng/ml GM-CSF as measured by flow cytometry. Bars are the mean ± SD normalized to the DMSO sample from n = 3 replicates. p -values calculated by unpaired Student’s t test between DMSO and other conditions. C , bar graph showing normalized GFP/mCherry ratio of CSF2RB reporter in TF1 Cas9 cells treated with 10 μM Chloroquine, 100 nM Bafilomycin A1, or a DMSO control for 4 h as measured by flow cytometry. Bars are the mean ± SD normalized to the DMSO sample from three biological replicates. p -values calculated by unpaired Student’s t test between DMSO and other conditions. D , volcano plot showing gene level analysis of CSF2RB reporter-based ubiquitin ligase–specific CRISPR screen in TF1 cells cultured in 5 ng/ml GM-CSF. Guide counts were collapsed to gene level (n = 4 guides/gene; two-sided empirical rank sum test statistics). E , volcano plot showing gene level analysis of CSF2RB reporter-based ubiquitin ligase–specific CRISPR screen in TF1 cells starved (0 min) and then stimulated with 5 ng/ml GM-CSF for 10 and 120 min. Guide counts were collapsed to gene level (n = 4 guides/gene; two-sided empirical rank-sum test statistics). F , volcano plot showing gene level analysis of Csf2rb reporter-based whole-genome CRISPR screen in 32D cells cultured in 0.01 ng/ml IL-3. Guide counts were collapsed to gene level (n = 4 guides/gene; two-sided empirical rank-sum test statistics). G , volcano plot of Pearson correlation coefficient and -log10( p -value) for linear correlation analysis of all gene CERES scores with STUB1 CERES scores in the Cancer Dependency Map (21Q4 public dataset). Inset shows scatterplot of CERES scores for STUB1 (horizontal axis) and CHIC2 (vertical axis).DMSO, dimethyl sulfoxide.

Article Snippet: Mm00655745_m1 , Csf2rb , Mouse , FAM-MGB , Thermo Fisher Scientific , 4331182.

Techniques: Ubiquitin Proteomics, CRISPR, Control, Flow Cytometry, Cell Culture

Journal: The Journal of Biological Chemistry

Article Title: A STUB1 ubiquitin ligase/CHIC2 protein complex negatively regulates the IL-3, IL-5, and GM-CSF cytokine receptor common β chain (CSF2RB) protein stability

doi: 10.1016/j.jbc.2022.102484

Figure Lengend Snippet: Stub1 and Chic2 KO lead to increased endogenous total and cell surface Csf2rb. A , Western blots of 32D Cas9 cells with sgNT, sgStub1-1, or sgStub1-2 for STUB1 and Vinculin. B , Western blots of 32D Cas9 cells with sgNT, sgChic2-1, or sgChic2-2 for Chic2 and Vinculin. C , Western blots of Csf2rb and Vinculin in 32D Cas9 cells with sgNT, sgStub1-1, or sgStub1-2 cultured in 5, 0.1, or 0.01 ng/ml IL-3 (S.E. = short exposure, L.E. = long exposure). Representative of three independent biological replicates with similar results. D , Western blots of Csf2rb and Vinculin in 32D Cas9 cells with sgNT, sgChic2-1, or sgChic2-2 cultured in 5, 0.1, or 0.01 ng/ml IL-3. Representative of three independent biological replicates with similar results. E , Western blots of Csf2rb and Vinculin in 32D Cas9 cells with sgNT, sgStub1-1, or sgStub1-2 treated with 10 μM cycloheximide (CHX) for 0, 2, 4, or 8 h. F , Western blots of Csf2rb and Vinculin in 32D Cas9 cells with sgNT, sgChic2-1, or sgChic2-2 treated with 10 μM cycloheximide (CHX) for 0, 2, 4, or 8 h. G , Western blots of Csf2rb, Stub1, Chic2, and Vinculin in 32D Cas9 cells with sgNT/sgNT, sgStub1-1/sgNT, sgNT/sgChic2-1, or sgStub1-1/sgChic2-2. H , bar graph showing ratio of mean GFP to mean mCherry signal of Csf2rb reporter in 32D cells with sgNT, sgChic2-1/2, or sgStub1-1/2 in 0.01 ng/ml IL-3 as measured by flow cytometry. Bars show GFP/mCherry ± SD from n = 3 replicates. I , bar graph showing median fluorescence intensity for anti-Csf2rb-PE (CD131) in 32D Cas9 cells with sgNT, sgChic2-1/2, or sgStub1-1/2 in 0.01 ng/m IL-3 as measured by flow cytometry. Bars show mean ± SD from n = 3 replicates. p -values calculated by unpaired Student’s t test between sgNT and other conditions at each cytokine concentration.

Article Snippet: Mm00655745_m1 , Csf2rb , Mouse , FAM-MGB , Thermo Fisher Scientific , 4331182.

Techniques: Western Blot, Cell Culture, Flow Cytometry, Fluorescence, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: A STUB1 ubiquitin ligase/CHIC2 protein complex negatively regulates the IL-3, IL-5, and GM-CSF cytokine receptor common β chain (CSF2RB) protein stability

doi: 10.1016/j.jbc.2022.102484

Figure Lengend Snippet: The Stub1/Chic2 complex interacts with Csf2rb and Chic2 and Stub1 KO reduce Csf2rb ubiquitination. A , Western blots of anti-V5 immunoprecipitation of STUB1-V5 and whole cell lysate for V5, Chic2, Csf2rb, and Vinculin from 32D Cas9 cells cultured in 0.01 ng/ml IL-3. B , Western blots of anti-V5 immunoprecipitation of V5-CHIC2 and whole cell lysate for V5, Stub1, Csf2rb, and Vinculin from 32D Cas9 cells cultured in 0.01 ng/ml IL-3. C , Western blots of anti-V5 immunoprecipitation of STUB1-V5 and whole cell lysate for V5, Csf2rb, and Vinculin from 32D Cas9 cells cultured in 5, 0.1, or 0.01 ng/ml IL-3. D , Western blots of anti-V5 immunoprecipitation of STUB1-V5 or STUB1 ΔTPR-V5 and whole cell lysate for V5, Csf2rb, Chic2, and Vinculin from 32D Cas9 cells cultured in 0.01 ng/ml IL-3. E , Western blots of anti-V5 immunoprecipitation of STUB1-V5 and whole cell lysate for V5, Csf2rb, Chic2, and Vinculin in 32D Cas9 cells with sgNT or sgChic2-1/2 cultured in 0.01 ng/ml IL-3. F , Western blots of anti-V5 immunoprecipitation of STUB1-V5 and whole cell lysate for V5, Csf2rb, and Vinculin from 32D Cas9 cells cultured in 5, 0.1, or 0.01 ng/ml IL-3. G , Western blots of anti-V5 immunoprecipitation of STUB1-V5 and whole cell lysate for V5, Csf2rb, Chic2, and Vinculin from 32D Cas9 cells cultured in 0.01 ng/ml IL-3 and treated with or without Ruxolitinib (100 nM) for 2 h.

Article Snippet: Mm00655745_m1 , Csf2rb , Mouse , FAM-MGB , Thermo Fisher Scientific , 4331182.

Techniques: Ubiquitin Proteomics, Western Blot, Immunoprecipitation, Cell Culture

Journal: The Journal of Biological Chemistry

Article Title: A STUB1 ubiquitin ligase/CHIC2 protein complex negatively regulates the IL-3, IL-5, and GM-CSF cytokine receptor common β chain (CSF2RB) protein stability

doi: 10.1016/j.jbc.2022.102484

Figure Lengend Snippet: Stub1 and Chic2 KO lead to deceased ubiquitination of Csf2rb and blocks the effect of a lysosomal acidification inhibitor on Csf2rb stability. A , Western blots of TUBE immunoprecipitation and whole cell lysate for Csf2rb, Chic2, Stub1, and Vinculin as a loading control in 32D Cas9 cells with sgNT, sgChic2-1/2, or sgStub1-1/2 cultured in 0.01 ng/ml IL-3 and treated with 1 μM Bafilomycin A1 and 10 μM MG132 for 4 h. B , Western blots of Csf2rb and Vinculin in 32D cas9 cells with sgNT, sgStub1-1, or sgChic2-1 treated with DMSO or bafilomycin A1 (100 nM) for 16 h. DMSO, dimethyl sulfoxide.

Article Snippet: Mm00655745_m1 , Csf2rb , Mouse , FAM-MGB , Thermo Fisher Scientific , 4331182.

Techniques: Ubiquitin Proteomics, Western Blot, Immunoprecipitation, Control, Cell Culture

Journal: The Journal of Biological Chemistry

Article Title: A STUB1 ubiquitin ligase/CHIC2 protein complex negatively regulates the IL-3, IL-5, and GM-CSF cytokine receptor common β chain (CSF2RB) protein stability

doi: 10.1016/j.jbc.2022.102484

Figure Lengend Snippet: Primary antibodies:

Article Snippet: Mm00655745_m1 , Csf2rb , Mouse , FAM-MGB , Thermo Fisher Scientific , 4331182.

Techniques: Control

Journal: The Journal of Biological Chemistry

Article Title: A STUB1 ubiquitin ligase/CHIC2 protein complex negatively regulates the IL-3, IL-5, and GM-CSF cytokine receptor common β chain (CSF2RB) protein stability

doi: 10.1016/j.jbc.2022.102484

Figure Lengend Snippet: RT-qPCR Primers

Article Snippet: Mm00655745_m1 , Csf2rb , Mouse , FAM-MGB , Thermo Fisher Scientific , 4331182.

Techniques:

Journal: Blood

Article Title: Cbfb/Runx1 repression-independent blockage of differentiation and accumulation of Csf2rb-expressing cells by Cbfb-MYH11

doi: 10.1182/blood-2009-06-227413

Figure Lengend Snippet: Expression of the genes uniquely deregulated in Cbfb + /MYH11 embryos in mouse and human leukemias

Article Snippet: Cells were stained with fluorescein isothiocyanate–, phycoerythrin-, and allophycocyanin (APC)–conjugated antibodies to Ter119, Csf2rb (CD131), c-Kit, annexin V, and bromodeoxyuridine (BrdU; BD PharMingen); APC–cyanin 7–conjugated c-Kit, phycoerythrin–cyanin 7 FcγR II/III, APC-conjugated CD34 (eBioscience); fluorescein isothiocyanate–conjugated Il1rl1 (MD Biosciences); and Pacific blue–conjugated ScaI (Biolegend).

Techniques: Expressing

Journal: Blood

Article Title: Cbfb/Runx1 repression-independent blockage of differentiation and accumulation of Csf2rb-expressing cells by Cbfb-MYH11

doi: 10.1182/blood-2009-06-227413

Figure Lengend Snippet: Csf2rb expression is related to Cbfb-MYH11–induced differentiation defects. (A) Representative FACS plots of Csf2rb and Ter119 staining in primitive blood cells from E10.5 embryos of the indicated genotypes. Percentage of cells in each gate is given. N ≥ 3 for all genotypes. (B) Representative FACS plots of Csf2rb and c-Kit staining in the primitive blood of E8.5 embryos of the indicated genotypes. N ≥ 3 for all genotypes.

Article Snippet: Cells were stained with fluorescein isothiocyanate–, phycoerythrin-, and allophycocyanin (APC)–conjugated antibodies to Ter119, Csf2rb (CD131), c-Kit, annexin V, and bromodeoxyuridine (BrdU; BD PharMingen); APC–cyanin 7–conjugated c-Kit, phycoerythrin–cyanin 7 FcγR II/III, APC-conjugated CD34 (eBioscience); fluorescein isothiocyanate–conjugated Il1rl1 (MD Biosciences); and Pacific blue–conjugated ScaI (Biolegend).

Techniques: Expressing, Staining

Journal: Blood

Article Title: Cbfb/Runx1 repression-independent blockage of differentiation and accumulation of Csf2rb-expressing cells by Cbfb-MYH11

doi: 10.1182/blood-2009-06-227413

Figure Lengend Snippet: Csf2rb, Il1rl1, and Gata2 are expressed in both mouse and human leukemic cells. (A) Representative FACS plots of Csf2rb and Il1rl1 staining in lineage-negative, c-Kit+ ScaI+ (LKS+) and c-Kit+, ScaI− (LKS−) bone marrow and spleen from nonleukemic Cbfb+/+ mice, and leukemic cells from the spleen of mice expressing a conditional allele of Cbfb-MYH11 (Cbfb+/56M, Mx1-Cre). N ≥ 3 for Cbfb+/56M, Mx1-Cre. Expression of Csf2rb (B) and Il1rl1 (C) in the peripheral blood of leukemic Cbfb+/56, Mx1 Cre+ and control littermate mice. The control group contained Cbfb+/+, Mx1-Cre−; Cbfb+/56M, Mx1-Cre−; and Cbfb+/+, Mx1-Cre+ mice. All mice were treated with pI:pC in the same way. (D) Western blot analysis of Gata2 expression in the peripheral blood of a nonleukemic Cbfb+/+ adult mouse, 2 different leukemic adult mice expressing Cbfb-MYH11, and the human inv(16) AML-derived ME-1 cells. The bottom panel shows α-tubulin expression using the same blot. (E) Reverse transcription–PCR analysis using primers specific for human CSF2RB in ME-1 cells. An arrow indicates the band of the expected size. (F) FACS plot of ME-1 cells stained for C-KIT and IL1RL1. Percentage of cells in each gate is given.

Article Snippet: Cells were stained with fluorescein isothiocyanate–, phycoerythrin-, and allophycocyanin (APC)–conjugated antibodies to Ter119, Csf2rb (CD131), c-Kit, annexin V, and bromodeoxyuridine (BrdU; BD PharMingen); APC–cyanin 7–conjugated c-Kit, phycoerythrin–cyanin 7 FcγR II/III, APC-conjugated CD34 (eBioscience); fluorescein isothiocyanate–conjugated Il1rl1 (MD Biosciences); and Pacific blue–conjugated ScaI (Biolegend).

Techniques: Staining, Expressing, Western Blot, Derivative Assay

Journal: Blood

Article Title: Cbfb/Runx1 repression-independent blockage of differentiation and accumulation of Csf2rb-expressing cells by Cbfb-MYH11

doi: 10.1182/blood-2009-06-227413

Figure Lengend Snippet: Cbfb-MYH11 expression results in an abnormal Csf2rb+ population with reduced progenitor activity. Representative FACS plots of c-Kit and ScaI (A), and Csf2rb (B) staining in lineage-depleted (lin−) bone marrow from mice of Cbfb+/56/M, Mx1-Cre+ and Cbfb+/+ mice at the indicated number of days after treatment with pI:pC. Percentage of cells in each gate is given. N ≥ 3 for all genotypes. (C) Representative FACS plots of c-Kit and ScaI staining of the Csf2rb− and Csf2rb+ populations from lin− bone marrow from Cbfb+/56M, Mx1-Cre+ mice 10 days after Cbfb-MYH11 induction. (D) Bar graphs of the relative total numbers of colonies seen from lin− bone marrow cells from Cbfb+/56M, Mx1-Cre+ mice 10 days after induction of Cbfb-MYH11 expression, sorted for Csf2rb expression and grown in culture for 12 days. N ≥ 3. *Statistically significant difference (P < .01). (E) FACS staining for Csf2rb of colonies derived from the Csf2rb− cells described in panel C. Percentage of Csf2rb+ cells is indicated. Error bars indicate SD within the indicated cell population.

Article Snippet: Cells were stained with fluorescein isothiocyanate–, phycoerythrin-, and allophycocyanin (APC)–conjugated antibodies to Ter119, Csf2rb (CD131), c-Kit, annexin V, and bromodeoxyuridine (BrdU; BD PharMingen); APC–cyanin 7–conjugated c-Kit, phycoerythrin–cyanin 7 FcγR II/III, APC-conjugated CD34 (eBioscience); fluorescein isothiocyanate–conjugated Il1rl1 (MD Biosciences); and Pacific blue–conjugated ScaI (Biolegend).

Techniques: Expressing, Activity Assay, Staining, Derivative Assay

Journal: Blood

Article Title: Cbfb/Runx1 repression-independent blockage of differentiation and accumulation of Csf2rb-expressing cells by Cbfb-MYH11

doi: 10.1182/blood-2009-06-227413

Figure Lengend Snippet: A transient population of Csf2rb+ cells is seen in the peripheral blood after Cbfb-MYH11 induction. (A) Line graphs showing percentage (%) of Csf2rb+ cells in the peripheral blood at the indicated number of weeks after pI:pC treatment in 3 representative mice. The line marked with circles represents a wild-type mouse after pI:pC treatment. The line marked with triangles represents a conditional Cbfb-MYH11 knockin mouse after pI:pC treatment that did not develop leukemia within the observed time period (7 weeks). The line marked with diamonds represents a conditional Cbfb-MYH11 knockin mouse after pI:pC treatment that spontaneously developed AML (without ENU). (B) Percentage of Csf2rb+ cells in a larger cohort of mice at the indicated number of weeks after pI:pC treatments. *Statistically significant difference between Cbfb+/56M, Mx1-Cre and control mice (P < .02). Wright-Giemsa staining of sorted Csf2rb+ peripheral blood cells from (C) a preleukemic Cbfb+/56M, Mx1-Cre mouse 2 weeks after Cbfb-MYH11 induction and (D) a leukemic Cbfb+/56M, Mx1-Cre mouse. Magnification, 1000×.

Article Snippet: Cells were stained with fluorescein isothiocyanate–, phycoerythrin-, and allophycocyanin (APC)–conjugated antibodies to Ter119, Csf2rb (CD131), c-Kit, annexin V, and bromodeoxyuridine (BrdU; BD PharMingen); APC–cyanin 7–conjugated c-Kit, phycoerythrin–cyanin 7 FcγR II/III, APC-conjugated CD34 (eBioscience); fluorescein isothiocyanate–conjugated Il1rl1 (MD Biosciences); and Pacific blue–conjugated ScaI (Biolegend).

Techniques: Knock-In, Staining

Journal: Blood

Article Title: Cbfb/Runx1 repression-independent blockage of differentiation and accumulation of Csf2rb-expressing cells by Cbfb-MYH11

doi: 10.1182/blood-2009-06-227413

Figure Lengend Snippet: Csf2rb−/lin− BM cells in mice expressing Cbfb-MYH11 are enriched for preleukemic progenitors and leukemia-initiating cells. (A) FACS staining of lineage-depleted bone marrow cells from ENU-treated, preleukemic Cbfb+/56M, Mx1-Cre mice 14 days after induction of Cbfb-MYH11. Cells were sorted for Csf2rb expression as indicated by the boxes, and transplanted into sublethally irradiated mice via retro-orbital injection. (B) Kaplan-Meier survival curves of mice that received a transplant of preleukemic Csf2rb− or Csf2rb+ cells. (C) FACS staining for the indicated differentiation markers in the Csf2rb− and Csf2rb+ leukemic spleen cells from a representative mouse that developed AML after transplantation of preleukemic Csf2rb− cells. (D) Western blot of CBFβ-SMMHC expression in the Csf2rb− and Csf2rb+ leukemic cells of 2 different recipient animals. (E) Kaplan-Meier survival curves of secondary transplant recipient mice that underwent transplantation as described in panel A with leukemic Csf2rb− and Csf2rb+ cells.

Article Snippet: Cells were stained with fluorescein isothiocyanate–, phycoerythrin-, and allophycocyanin (APC)–conjugated antibodies to Ter119, Csf2rb (CD131), c-Kit, annexin V, and bromodeoxyuridine (BrdU; BD PharMingen); APC–cyanin 7–conjugated c-Kit, phycoerythrin–cyanin 7 FcγR II/III, APC-conjugated CD34 (eBioscience); fluorescein isothiocyanate–conjugated Il1rl1 (MD Biosciences); and Pacific blue–conjugated ScaI (Biolegend).

Techniques: Expressing, Staining, Irradiation, Injection, Transplantation Assay, Western Blot

Journal: Blood

Article Title: Cbfb/Runx1 repression-independent blockage of differentiation and accumulation of Csf2rb-expressing cells by Cbfb-MYH11

doi: 10.1182/blood-2009-06-227413

Figure Lengend Snippet: Model of Cbfb-MYH11–induced aberrant differentiation and leukemia initiation. We propose that expression of Cbfb-MYH11 in HSCs or early progenitors causes an abnormal and incomplete differentiation that culminates in a Csf2rb+ cell population. Our results indicate that by the time the Cbfb-MYH11–expressing cells have reached the Csf2rb+ stage, they are no longer capable of initiating leukemia.

Article Snippet: Cells were stained with fluorescein isothiocyanate–, phycoerythrin-, and allophycocyanin (APC)–conjugated antibodies to Ter119, Csf2rb (CD131), c-Kit, annexin V, and bromodeoxyuridine (BrdU; BD PharMingen); APC–cyanin 7–conjugated c-Kit, phycoerythrin–cyanin 7 FcγR II/III, APC-conjugated CD34 (eBioscience); fluorescein isothiocyanate–conjugated Il1rl1 (MD Biosciences); and Pacific blue–conjugated ScaI (Biolegend).

Techniques: Expressing

Journal: International Journal of Molecular Sciences

Article Title: Negative Regulation of the Differentiation of Flk2 − CD34 − LSK Hematopoietic Stem Cells by EKLF/KLF1

doi: 10.3390/ijms21228448

Figure Lengend Snippet: Regulation of Flk2-CD34-HSC differentiation by EKLF ( A ) RT-qPCR analysis of RNAs isolated from different types of mouse hematopoietic stem cells and progenitors, including LT-HSCs, MPPs, CMPs, GMPs, and MEPs purified from E14.5 mouse fetal liver and Flk2-CD34-HSCs from bone marrow (BM Flk2-CD34-HSC) by FACS. The relative levels of Eklf mRNA in all of the progenitor cells are compared to that of mouse erythroleukemia (MEL) cells in the left panel, with the level in MEP set as 1. The comparative RT-qPCR analyses of Eklf mRNA in Flk2-CD34-HSCs and MPPs of E14.5 fetal livers from WT and KO ( Eklf −/− ) embryos are shown in the right panel. ( B ) RT-qPCR analysis of the mRNA levels of Csf2rb , Stat1 , and Stat2 in purified CMPs, GMPs, and MPPs. Five biological replicates were analyzed for each type of cells. Each bar represents mean ± standard deviation. * p < 0.05, ** p < 0.01. ( C ) Relative levels of Csf2rb mRNA in Flk2-CD34-HSC purified from WT and KO E14.5 mouse fetal livers were analyzed by RT-qPCR. n ≥ 5. ** p < 0.01. ( D ) Representative immuno-fluorescence staining patterns of the expression patterns of EKLF and CSF2RB in Flk2-CD34-HSCs (WT) and Flk2-CD34-HSCs (KO). DAPI (49-6-diamidino-2-phenylindole) is the nucleus marker. Note the lack of EKLF signal in Flk2-CD34-HSCs (KO). Three biological replicates were analyzed. The diameters of LT-HSCs range from 5–10 μm. EKLF signal present in the nucleus (white arrow). ( E ) Colony-forming cell (CFC) assay was performed on Flk2-CD34-HSCs (WT) and Flk2-CD34-HSCs (KO) purified (>90%) from E14.5 fetal liver cells by flow cytometry. n ≥ 4. *** p < 0.001. ( F ) Top, map of Eklf transcript with 3 exons. Middle, Eklf exon 2 as the target of gDNA. Bottom, comparison of the genomic sequence of the CRISPR/Cas9-edited Eklf exon 2 region in ESC (Del) to the wild-type sequence in ESC (WT). The Eklf -F and Eklf -R PCR primers used for validating the sequences are indicated. Note the presence of 10 bp deletion in ESC(Del) that causes a frame shift and consequent pre-mature termination TGA codon. ( G ) CFC assay of embryoid body (EB) cells derived from wild-type ESC(WT) and from CRISPR/Cas9-edited ESC, i.e., ESC(Del), respectively. n = 3. ** p < 0.01.

Article Snippet: Flk2-CD34-HSCs purified by flow sorting, as described above, were suspended and fixed by 1% paraformaldehyde on 4-well culture slide (Millipore Millicell EZ SLIDE), permeabilized with 0.1% ( v / v ) Triton X-100, and stained with mouse anti-mouse CSF2RB (GeneTex, Irvine, CA, USA) or home-made rabbit anti-mouse EKLF [ ].

Techniques: Quantitative RT-PCR, Isolation, Purification, Standard Deviation, Fluorescence, Staining, Expressing, Marker, Hematopoietic Colony Assay, Flow Cytometry, Comparison, Sequencing, CRISPR, Derivative Assay

Journal: International Journal of Molecular Sciences

Article Title: Negative Regulation of the Differentiation of Flk2 − CD34 − LSK Hematopoietic Stem Cells by EKLF/KLF1

doi: 10.3390/ijms21228448

Figure Lengend Snippet: ( A ) FACS analysis of BMMNCs, Lin − Sca1 + c-Kit + (LSK) cells, and Flk2-CD34-HSCs in the bone marrow of recipient mice after serial transplantation. The bone marrow cells were stained with the appropriate antibodies and analyzed by FACS. Statistical analysis of the FACS data. The percentages of BMMNCs, ratios of LSK cells/BMMNCs, and ratios of Flk2-CD34-HSCs/BMMNCs in the bone marrows of primary, secondary, and tertiary recipient mice, as deduced from the FACS data, are shown in the histograms (primary transplantation, n = 3; secondary transplantation, n = 4; tertiary transplantation, n = 8). * p < 0.05, ** p < 0.01 ( B ) A simple model showing the negative regulatory roles of EKLF in the homeostasis of Flk2-CD34-HSCs. The factor restricts the self-renewal capability of Flk2-CD34-HSCs. It also acts as a repressor to prevent superfluous Csf2rb expression and consequent over-differentiation of Flk2-CD34-HSCs. For more details, see text.

Article Snippet: Flk2-CD34-HSCs purified by flow sorting, as described above, were suspended and fixed by 1% paraformaldehyde on 4-well culture slide (Millipore Millicell EZ SLIDE), permeabilized with 0.1% ( v / v ) Triton X-100, and stained with mouse anti-mouse CSF2RB (GeneTex, Irvine, CA, USA) or home-made rabbit anti-mouse EKLF [ ].

Techniques: Transplantation Assay, Staining, Expressing