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Miltenyi Biotec cd11b positive cells
Tyrosine hydroxylase staining of brown adipose tissue macrophages. ( a ) Representative FACS analysis of BAT macrophages isolated from pan-tdTomato, WT and Th Cre :tdTomato fl/fl mice ( n = 2 each genotype), either from mice housed at 22°C or at 4°C for 8 h; (top) macrophage gating strategy on <t>CD11b,</t> F4/80 and CD14 expressing cells. ( b ) Representative histology of BAT taken from Cx3cr1 cre :r26- YFP animals in 22°C, stained for TH (red) and YFP (green) ( n = 2) (scale bar: 50 µm). ( c ) Representative 2-photon live imaging of BAT taken from Th Cre :tdTomato fl/fl :Cx3cr1 gfp mice at 22°C ( n = 5) (scale bar: 50 µm). ( d ) Integrative Genomics Viewer (IGV) plots of RNA sequencing data of the TH locus of macrophages isolated from brain, BAT, spleen, liver, peritoneum, large and small intestine (Li, SI) under steady state; data are from 21 , except for BAT macrophages. Displayed results in panel d were performed in technical duplicates.
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Images

1) Product Images from "Alternatively activated macrophages do not synthesize catecholamines or contribute to adipose tissue adaptive thermogenesis"

Article Title: Alternatively activated macrophages do not synthesize catecholamines or contribute to adipose tissue adaptive thermogenesis

Journal: Nature medicine

doi: 10.1038/nm.4316

Tyrosine hydroxylase staining of brown adipose tissue macrophages. ( a ) Representative FACS analysis of BAT macrophages isolated from pan-tdTomato, WT and Th Cre :tdTomato fl/fl mice ( n = 2 each genotype), either from mice housed at 22°C or at 4°C for 8 h; (top) macrophage gating strategy on CD11b, F4/80 and CD14 expressing cells. ( b ) Representative histology of BAT taken from Cx3cr1 cre :r26- YFP animals in 22°C, stained for TH (red) and YFP (green) ( n = 2) (scale bar: 50 µm). ( c ) Representative 2-photon live imaging of BAT taken from Th Cre :tdTomato fl/fl :Cx3cr1 gfp mice at 22°C ( n = 5) (scale bar: 50 µm). ( d ) Integrative Genomics Viewer (IGV) plots of RNA sequencing data of the TH locus of macrophages isolated from brain, BAT, spleen, liver, peritoneum, large and small intestine (Li, SI) under steady state; data are from 21 , except for BAT macrophages. Displayed results in panel d were performed in technical duplicates.
Figure Legend Snippet: Tyrosine hydroxylase staining of brown adipose tissue macrophages. ( a ) Representative FACS analysis of BAT macrophages isolated from pan-tdTomato, WT and Th Cre :tdTomato fl/fl mice ( n = 2 each genotype), either from mice housed at 22°C or at 4°C for 8 h; (top) macrophage gating strategy on CD11b, F4/80 and CD14 expressing cells. ( b ) Representative histology of BAT taken from Cx3cr1 cre :r26- YFP animals in 22°C, stained for TH (red) and YFP (green) ( n = 2) (scale bar: 50 µm). ( c ) Representative 2-photon live imaging of BAT taken from Th Cre :tdTomato fl/fl :Cx3cr1 gfp mice at 22°C ( n = 5) (scale bar: 50 µm). ( d ) Integrative Genomics Viewer (IGV) plots of RNA sequencing data of the TH locus of macrophages isolated from brain, BAT, spleen, liver, peritoneum, large and small intestine (Li, SI) under steady state; data are from 21 , except for BAT macrophages. Displayed results in panel d were performed in technical duplicates.

Techniques Used: Staining, FACS, Isolation, Mouse Assay, Expressing, Imaging, RNA Sequencing Assay

2) Product Images from "Targeting glutaminolysis has antileukemic activity in acute myeloid leukemia and synergizes with BCL-2 inhibition"

Article Title: Targeting glutaminolysis has antileukemic activity in acute myeloid leukemia and synergizes with BCL-2 inhibition

Journal: Blood

doi: 10.1182/blood-2015-01-621870

GAC protein is prominently expressed in AML and modulates the OCR. (A) Analysis of human leukemic cell lines. AML cells from 8 patients and normal CD34 + HPCs from 4 healthy donors were analyzed by western blotting using anti-GLS1, anti-GLS2, and anti-ACTIN antibodies. (B) MOLM-14 (left) and OCI-AML2 (right) cells were transfected with a lentiviral vector expressing a doxycycline-inducible GLS1 shRNA (#5) construct. Stably infected cell lines were established by puromycin selection. After 2 days of doxycycline exposure, the OCR was measured using a Seahorse XF96 extracellular flux analyzer under both basal conditions and after the addition of CCCP and antimycin, as indicated. The inhibition of GAC expression was controlled in western blots using anti-GAC antibody. Histograms show data that are representative of 3 independent experiments. (C) OCI-AML2 cells were cultured with or without CB-839 (1 µM), BPTES (10 µM), or compound 968 (10 µM) for 6 hours, and the OCR was measured using a Seahorse XF96 extracellular flux analyzer. Histograms show data that are representative of 3 independent experiments. (D) OCI-AML2 cells were cultured with or without CB-839 (1 µM), BPTES (10 µM), or compound 968 (10 µM) for 4 hours, and 5 × 10 6 cells were washed twice in cold PBS; the pellet was frozen and each indicted metabolite was measured. (E) OCI-AML2 cells were cultured with or without CB-839 (1 µM) and αKG (5 mM) for 6 h, and the OCR was measured. Histograms show data that are representative of 3 independent experiments. (F) OCI-AML2 cells were transfected with a lentiviral vector expressing a doxycycline-inducible V5-tagged GAC WT or GAC K320A construct. Stably infected cell lines were established by puromycin selection. After 6-hour doxycycline exposure with or without CB-839, the OCR was measured under both basal conditions and after the addition of CCCP and antimycin, as indicated. Histograms show data that are representative of 3 independent experiments (G) OCI-AML2 cells stably infected with GAC WT or GAC K320A were cultured for 6 hours with or without doxycycline or CB-839 (1 µM) and were analyzed by western blotting using anti-GAC and anti-ACTIN antibodies. * P
Figure Legend Snippet: GAC protein is prominently expressed in AML and modulates the OCR. (A) Analysis of human leukemic cell lines. AML cells from 8 patients and normal CD34 + HPCs from 4 healthy donors were analyzed by western blotting using anti-GLS1, anti-GLS2, and anti-ACTIN antibodies. (B) MOLM-14 (left) and OCI-AML2 (right) cells were transfected with a lentiviral vector expressing a doxycycline-inducible GLS1 shRNA (#5) construct. Stably infected cell lines were established by puromycin selection. After 2 days of doxycycline exposure, the OCR was measured using a Seahorse XF96 extracellular flux analyzer under both basal conditions and after the addition of CCCP and antimycin, as indicated. The inhibition of GAC expression was controlled in western blots using anti-GAC antibody. Histograms show data that are representative of 3 independent experiments. (C) OCI-AML2 cells were cultured with or without CB-839 (1 µM), BPTES (10 µM), or compound 968 (10 µM) for 6 hours, and the OCR was measured using a Seahorse XF96 extracellular flux analyzer. Histograms show data that are representative of 3 independent experiments. (D) OCI-AML2 cells were cultured with or without CB-839 (1 µM), BPTES (10 µM), or compound 968 (10 µM) for 4 hours, and 5 × 10 6 cells were washed twice in cold PBS; the pellet was frozen and each indicted metabolite was measured. (E) OCI-AML2 cells were cultured with or without CB-839 (1 µM) and αKG (5 mM) for 6 h, and the OCR was measured. Histograms show data that are representative of 3 independent experiments. (F) OCI-AML2 cells were transfected with a lentiviral vector expressing a doxycycline-inducible V5-tagged GAC WT or GAC K320A construct. Stably infected cell lines were established by puromycin selection. After 6-hour doxycycline exposure with or without CB-839, the OCR was measured under both basal conditions and after the addition of CCCP and antimycin, as indicated. Histograms show data that are representative of 3 independent experiments (G) OCI-AML2 cells stably infected with GAC WT or GAC K320A were cultured for 6 hours with or without doxycycline or CB-839 (1 µM) and were analyzed by western blotting using anti-GAC and anti-ACTIN antibodies. * P

Techniques Used: Western Blot, Transfection, Plasmid Preparation, Expressing, shRNA, Construct, Stable Transfection, Infection, Selection, Inhibition, Cell Culture

Targeting glutaminase activity inhibits AML cell proliferation. (A) OCI-AML2 and MOLM-14 shGLS1#5 leukemic cells were seeded at 5 × 10 5 cells/mL with or without doxycycline; viable cells were counted manually following trypan blue staining on days 3, 5, and 7. Each experiment was performed independently ≥3 times. (B) OCI-AML2, MV4-11, MOLM-14, HL-60, and OCI-AML3 cells transfected with shGLS1#5 or #7 were seeded at 5 × 10 5 cells/mL with or without doxycycline; viable cells were counted following trypan blue staining on days 3, 5, and 7. Each experiment was performed independently ≥3 times. (C) OCI-AML2 and MOLM-14 shGLS1#5 leukemic cells were seeded at 5 × 10 5 cells/mL with or without doxycycline, and αKG (5 mM) and viable cells were counted following trypan blue staining on day 7. (D) A total of 10 cell lines were seeded at 5 × 10 5 cells/mL with or without CB-839 (1 µM); viable cells were counted following trypan blue staining on days 3, 5, and 7. (E) OCI-AML2 cells were seeded at 5 × 10 5 cells/mL with or without CB-839 (1 µM) and αKG (5 mM); viable cells were counted following trypan blue staining on days 3, 5, and 7. (F) CD34 + HPCs from 4 healthy donors were seeded at 5 × 10 5 cells/mL with and without CB-839 (1 µM); viable cells were counted following trypan blue staining on days 2, 3, 4, and 5. * P
Figure Legend Snippet: Targeting glutaminase activity inhibits AML cell proliferation. (A) OCI-AML2 and MOLM-14 shGLS1#5 leukemic cells were seeded at 5 × 10 5 cells/mL with or without doxycycline; viable cells were counted manually following trypan blue staining on days 3, 5, and 7. Each experiment was performed independently ≥3 times. (B) OCI-AML2, MV4-11, MOLM-14, HL-60, and OCI-AML3 cells transfected with shGLS1#5 or #7 were seeded at 5 × 10 5 cells/mL with or without doxycycline; viable cells were counted following trypan blue staining on days 3, 5, and 7. Each experiment was performed independently ≥3 times. (C) OCI-AML2 and MOLM-14 shGLS1#5 leukemic cells were seeded at 5 × 10 5 cells/mL with or without doxycycline, and αKG (5 mM) and viable cells were counted following trypan blue staining on day 7. (D) A total of 10 cell lines were seeded at 5 × 10 5 cells/mL with or without CB-839 (1 µM); viable cells were counted following trypan blue staining on days 3, 5, and 7. (E) OCI-AML2 cells were seeded at 5 × 10 5 cells/mL with or without CB-839 (1 µM) and αKG (5 mM); viable cells were counted following trypan blue staining on days 3, 5, and 7. (F) CD34 + HPCs from 4 healthy donors were seeded at 5 × 10 5 cells/mL with and without CB-839 (1 µM); viable cells were counted following trypan blue staining on days 2, 3, 4, and 5. * P

Techniques Used: Activity Assay, Staining, Transfection

GLS1 controls AML survival by modulating the TCA cycle. (A) ShGLS1#5 was induced with doxycycline for 4 days in OCI-AML2, HL-60, MV4-11, MOLM-14, and OCI-AML3 leukemic cells, and apoptosis was evaluated based on Annexin-V binding. (B) CD34 + HPCs from 3 healthy donors were transfected with a lentiviral vector that expressed a noninducible GLS1 shRNA (#5), and transfected cells were selected with puromycin for 2 days; 3 days later, apoptosis was evaluated based on Annexin-V staining, and protein extracts were immunobloted with anti-GAC and anti-ACTIN antibodies. (C) MOLM-14 shGLS1 cells, MOLM-14 shCTR cells (9 mice each), and OCI-AML2 shGLS1 cells or OCI-AML2 shCTR cells (9 mice each) were intravenously injected into NSG mice (2 × 10 6 cells per mouse). Mice were treated with doxycycline by oral gavage. The Kaplan-Meier survival curves of mice treated with doxycycline are shown for both cell lines. (D) The spleens of the mice injected with the OCI-AML2 shGLS1 and OCI-AML2 shCTR cell lines were measured. (E) A total of 11 cell lines were cultured for 4 days with or without CB-839 (1 µM), and apoptosis was evaluated based on Annexin-V staining. (F) OCI-AML2 and HL-60 cells were cultured for 4 days with or without CB-839 (1 µM), and dmαKG (5 mM) and apoptosis was evaluated based on Annexin-V staining. (G) OCI-AML2 cells that were stably infected with GAC WT or GAC K320A were cultured for 2 days with or without doxycycline or CB-839 (1 µM). Apoptosis was evaluated based on Annexin-V staining. (H) AML samples from 10 patients and CD34 + HPCs from 6 healthy donors were cultured for 4 days with or without CB-839 (1 µM). Apoptosis was evaluated based on Annexin-V staining. * P
Figure Legend Snippet: GLS1 controls AML survival by modulating the TCA cycle. (A) ShGLS1#5 was induced with doxycycline for 4 days in OCI-AML2, HL-60, MV4-11, MOLM-14, and OCI-AML3 leukemic cells, and apoptosis was evaluated based on Annexin-V binding. (B) CD34 + HPCs from 3 healthy donors were transfected with a lentiviral vector that expressed a noninducible GLS1 shRNA (#5), and transfected cells were selected with puromycin for 2 days; 3 days later, apoptosis was evaluated based on Annexin-V staining, and protein extracts were immunobloted with anti-GAC and anti-ACTIN antibodies. (C) MOLM-14 shGLS1 cells, MOLM-14 shCTR cells (9 mice each), and OCI-AML2 shGLS1 cells or OCI-AML2 shCTR cells (9 mice each) were intravenously injected into NSG mice (2 × 10 6 cells per mouse). Mice were treated with doxycycline by oral gavage. The Kaplan-Meier survival curves of mice treated with doxycycline are shown for both cell lines. (D) The spleens of the mice injected with the OCI-AML2 shGLS1 and OCI-AML2 shCTR cell lines were measured. (E) A total of 11 cell lines were cultured for 4 days with or without CB-839 (1 µM), and apoptosis was evaluated based on Annexin-V staining. (F) OCI-AML2 and HL-60 cells were cultured for 4 days with or without CB-839 (1 µM), and dmαKG (5 mM) and apoptosis was evaluated based on Annexin-V staining. (G) OCI-AML2 cells that were stably infected with GAC WT or GAC K320A were cultured for 2 days with or without doxycycline or CB-839 (1 µM). Apoptosis was evaluated based on Annexin-V staining. (H) AML samples from 10 patients and CD34 + HPCs from 6 healthy donors were cultured for 4 days with or without CB-839 (1 µM). Apoptosis was evaluated based on Annexin-V staining. * P

Techniques Used: Binding Assay, Transfection, Plasmid Preparation, shRNA, Staining, Mouse Assay, Injection, Cell Culture, Stable Transfection, Infection

3) Product Images from "Mild heat treatment primes human CD34+ cord blood cells for migration towards SDF-1α and enhances engraftment in an NSG mouse model"

Article Title: Mild heat treatment primes human CD34+ cord blood cells for migration towards SDF-1α and enhances engraftment in an NSG mouse model

Journal: Stem cells (Dayton, Ohio)

doi: 10.1002/stem.1988

Heat significantly increases the percent of CD34 + CB and Mo7e cells with aggregated lipid rafts and CXCR4 receptors on their cell surface
Figure Legend Snippet: Heat significantly increases the percent of CD34 + CB and Mo7e cells with aggregated lipid rafts and CXCR4 receptors on their cell surface

Techniques Used:

Heat treatment of human CD34 + CB cells prior to transplantation into NSG significantly increases the percent human CD45 + cell engraftment
Figure Legend Snippet: Heat treatment of human CD34 + CB cells prior to transplantation into NSG significantly increases the percent human CD45 + cell engraftment

Techniques Used: Transplantation Assay

Heating CD34 + CB and Mo7e cells significantly enhances their migration towards an SDF-1 gradient
Figure Legend Snippet: Heating CD34 + CB and Mo7e cells significantly enhances their migration towards an SDF-1 gradient

Techniques Used: Migration

The enhancement in chemotaxis seen following heating of CD34 + CB cells is dependent upon lipid raft aggregation
Figure Legend Snippet: The enhancement in chemotaxis seen following heating of CD34 + CB cells is dependent upon lipid raft aggregation

Techniques Used: Chemotaxis Assay

Activation of Rac1 is essential for the enhancement in migration seen following heat treatment of CD34 + CB cells
Figure Legend Snippet: Activation of Rac1 is essential for the enhancement in migration seen following heat treatment of CD34 + CB cells

Techniques Used: Activation Assay, Migration

4) Product Images from "Control of Adipose Tissue Inflammation Through TRB1"

Article Title: Control of Adipose Tissue Inflammation Through TRB1

Journal: Diabetes

doi: 10.2337/db09-1537

TRB1 expression is under the control of cytokine signaling in an adipocyte-autonomous manner. A : Quantitative PCR analysis of TRB1 mRNA levels in WAT depots, mature adipocytes after separation from SVFs, SVF- or CD11b + -enriched cellular fractions from WAT depots. B and C : Quantitative PCR analysis of TRB1 mRNA levels in primary adipocytes derived from the stromal-vascular WAT fractions of wild-type (wt) and TLR4 ( B ) or p50 ( C ) knockout (ko) mice. Cells were treated with LPS and LPS-conditioned (conditioned medium [CM]) or non–LPS-conditioned (control [M]) macrophage supernatant as indicated. D and E : Quantitative PCR analysis of TRB1 mRNA levels in mature adipocytes derived from 3T3–L1 preadipocytes ( D ) or the SVF of wild-type mice ( E ). Cells were pretreated with inhibitors against p38 (SB202190), the ERK (PD98059), and NF-κB (parthenolide) pathways and subsequently stimulated with LPS-conditioned (conditioned medium [CM]) or non–LPS-conditioned (control [M]) macrophage supernatant as indicated (means ± SE, n = 5). * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001. n.s., non significant.
Figure Legend Snippet: TRB1 expression is under the control of cytokine signaling in an adipocyte-autonomous manner. A : Quantitative PCR analysis of TRB1 mRNA levels in WAT depots, mature adipocytes after separation from SVFs, SVF- or CD11b + -enriched cellular fractions from WAT depots. B and C : Quantitative PCR analysis of TRB1 mRNA levels in primary adipocytes derived from the stromal-vascular WAT fractions of wild-type (wt) and TLR4 ( B ) or p50 ( C ) knockout (ko) mice. Cells were treated with LPS and LPS-conditioned (conditioned medium [CM]) or non–LPS-conditioned (control [M]) macrophage supernatant as indicated. D and E : Quantitative PCR analysis of TRB1 mRNA levels in mature adipocytes derived from 3T3–L1 preadipocytes ( D ) or the SVF of wild-type mice ( E ). Cells were pretreated with inhibitors against p38 (SB202190), the ERK (PD98059), and NF-κB (parthenolide) pathways and subsequently stimulated with LPS-conditioned (conditioned medium [CM]) or non–LPS-conditioned (control [M]) macrophage supernatant as indicated (means ± SE, n = 5). * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001. n.s., non significant.

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Derivative Assay, Knock-Out, Mouse Assay

5) Product Images from "Alternatively activated macrophages do not synthesize catecholamines or contribute to adipose tissue adaptive thermogenesis"

Article Title: Alternatively activated macrophages do not synthesize catecholamines or contribute to adipose tissue adaptive thermogenesis

Journal: Nature medicine

doi: 10.1038/nm.4316

Tyrosine hydroxylase staining of brown adipose tissue macrophages. ( a ) Representative FACS analysis of BAT macrophages isolated from pan-tdTomato, WT and Th Cre :tdTomato fl/fl mice ( n = 2 each genotype), either from mice housed at 22°C or at 4°C for 8 h; (top) macrophage gating strategy on CD11b, F4/80 and CD14 expressing cells. ( b ) Representative histology of BAT taken from Cx3cr1 cre :r26- YFP animals in 22°C, stained for TH (red) and YFP (green) ( n = 2) (scale bar: 50 µm). ( c ) Representative 2-photon live imaging of BAT taken from Th Cre :tdTomato fl/fl :Cx3cr1 gfp mice at 22°C ( n = 5) (scale bar: 50 µm). ( d ) Integrative Genomics Viewer (IGV) plots of RNA sequencing data of the TH locus of macrophages isolated from brain, BAT, spleen, liver, peritoneum, large and small intestine (Li, SI) under steady state; data are from 21 , except for BAT macrophages. Displayed results in panel d were performed in technical duplicates.
Figure Legend Snippet: Tyrosine hydroxylase staining of brown adipose tissue macrophages. ( a ) Representative FACS analysis of BAT macrophages isolated from pan-tdTomato, WT and Th Cre :tdTomato fl/fl mice ( n = 2 each genotype), either from mice housed at 22°C or at 4°C for 8 h; (top) macrophage gating strategy on CD11b, F4/80 and CD14 expressing cells. ( b ) Representative histology of BAT taken from Cx3cr1 cre :r26- YFP animals in 22°C, stained for TH (red) and YFP (green) ( n = 2) (scale bar: 50 µm). ( c ) Representative 2-photon live imaging of BAT taken from Th Cre :tdTomato fl/fl :Cx3cr1 gfp mice at 22°C ( n = 5) (scale bar: 50 µm). ( d ) Integrative Genomics Viewer (IGV) plots of RNA sequencing data of the TH locus of macrophages isolated from brain, BAT, spleen, liver, peritoneum, large and small intestine (Li, SI) under steady state; data are from 21 , except for BAT macrophages. Displayed results in panel d were performed in technical duplicates.

Techniques Used: Staining, FACS, Isolation, Mouse Assay, Expressing, Imaging, RNA Sequencing Assay

6) Product Images from "Targeting glutaminolysis has antileukemic activity in acute myeloid leukemia and synergizes with BCL-2 inhibition"

Article Title: Targeting glutaminolysis has antileukemic activity in acute myeloid leukemia and synergizes with BCL-2 inhibition

Journal: Blood

doi: 10.1182/blood-2015-01-621870

GAC protein is prominently expressed in AML and modulates the OCR. (A) Analysis of human leukemic cell lines. AML cells from 8 patients and normal CD34 + HPCs from 4 healthy donors were analyzed by western blotting using anti-GLS1, anti-GLS2, and anti-ACTIN antibodies. (B) MOLM-14 (left) and OCI-AML2 (right) cells were transfected with a lentiviral vector expressing a doxycycline-inducible GLS1 shRNA (#5) construct. Stably infected cell lines were established by puromycin selection. After 2 days of doxycycline exposure, the OCR was measured using a Seahorse XF96 extracellular flux analyzer under both basal conditions and after the addition of CCCP and antimycin, as indicated. The inhibition of GAC expression was controlled in western blots using anti-GAC antibody. Histograms show data that are representative of 3 independent experiments. (C) OCI-AML2 cells were cultured with or without CB-839 (1 µM), BPTES (10 µM), or compound 968 (10 µM) for 6 hours, and the OCR was measured using a Seahorse XF96 extracellular flux analyzer. Histograms show data that are representative of 3 independent experiments. (D) OCI-AML2 cells were cultured with or without CB-839 (1 µM), BPTES (10 µM), or compound 968 (10 µM) for 4 hours, and 5 × 10 6 cells were washed twice in cold PBS; the pellet was frozen and each indicted metabolite was measured. (E) OCI-AML2 cells were cultured with or without CB-839 (1 µM) and αKG (5 mM) for 6 h, and the OCR was measured. Histograms show data that are representative of 3 independent experiments. (F) OCI-AML2 cells were transfected with a lentiviral vector expressing a doxycycline-inducible V5-tagged GAC WT or GAC K320A construct. Stably infected cell lines were established by puromycin selection. After 6-hour doxycycline exposure with or without CB-839, the OCR was measured under both basal conditions and after the addition of CCCP and antimycin, as indicated. Histograms show data that are representative of 3 independent experiments (G) OCI-AML2 cells stably infected with GAC WT or GAC K320A were cultured for 6 hours with or without doxycycline or CB-839 (1 µM) and were analyzed by western blotting using anti-GAC and anti-ACTIN antibodies. * P
Figure Legend Snippet: GAC protein is prominently expressed in AML and modulates the OCR. (A) Analysis of human leukemic cell lines. AML cells from 8 patients and normal CD34 + HPCs from 4 healthy donors were analyzed by western blotting using anti-GLS1, anti-GLS2, and anti-ACTIN antibodies. (B) MOLM-14 (left) and OCI-AML2 (right) cells were transfected with a lentiviral vector expressing a doxycycline-inducible GLS1 shRNA (#5) construct. Stably infected cell lines were established by puromycin selection. After 2 days of doxycycline exposure, the OCR was measured using a Seahorse XF96 extracellular flux analyzer under both basal conditions and after the addition of CCCP and antimycin, as indicated. The inhibition of GAC expression was controlled in western blots using anti-GAC antibody. Histograms show data that are representative of 3 independent experiments. (C) OCI-AML2 cells were cultured with or without CB-839 (1 µM), BPTES (10 µM), or compound 968 (10 µM) for 6 hours, and the OCR was measured using a Seahorse XF96 extracellular flux analyzer. Histograms show data that are representative of 3 independent experiments. (D) OCI-AML2 cells were cultured with or without CB-839 (1 µM), BPTES (10 µM), or compound 968 (10 µM) for 4 hours, and 5 × 10 6 cells were washed twice in cold PBS; the pellet was frozen and each indicted metabolite was measured. (E) OCI-AML2 cells were cultured with or without CB-839 (1 µM) and αKG (5 mM) for 6 h, and the OCR was measured. Histograms show data that are representative of 3 independent experiments. (F) OCI-AML2 cells were transfected with a lentiviral vector expressing a doxycycline-inducible V5-tagged GAC WT or GAC K320A construct. Stably infected cell lines were established by puromycin selection. After 6-hour doxycycline exposure with or without CB-839, the OCR was measured under both basal conditions and after the addition of CCCP and antimycin, as indicated. Histograms show data that are representative of 3 independent experiments (G) OCI-AML2 cells stably infected with GAC WT or GAC K320A were cultured for 6 hours with or without doxycycline or CB-839 (1 µM) and were analyzed by western blotting using anti-GAC and anti-ACTIN antibodies. * P

Techniques Used: Western Blot, Transfection, Plasmid Preparation, Expressing, shRNA, Construct, Stable Transfection, Infection, Selection, Inhibition, Cell Culture

Targeting glutaminase activity inhibits AML cell proliferation. (A) OCI-AML2 and MOLM-14 shGLS1#5 leukemic cells were seeded at 5 × 10 5 cells/mL with or without doxycycline; viable cells were counted manually following trypan blue staining on days 3, 5, and 7. Each experiment was performed independently ≥3 times. (B) OCI-AML2, MV4-11, MOLM-14, HL-60, and OCI-AML3 cells transfected with shGLS1#5 or #7 were seeded at 5 × 10 5 cells/mL with or without doxycycline; viable cells were counted following trypan blue staining on days 3, 5, and 7. Each experiment was performed independently ≥3 times. (C) OCI-AML2 and MOLM-14 shGLS1#5 leukemic cells were seeded at 5 × 10 5 cells/mL with or without doxycycline, and αKG (5 mM) and viable cells were counted following trypan blue staining on day 7. (D) A total of 10 cell lines were seeded at 5 × 10 5 cells/mL with or without CB-839 (1 µM); viable cells were counted following trypan blue staining on days 3, 5, and 7. (E) OCI-AML2 cells were seeded at 5 × 10 5 cells/mL with or without CB-839 (1 µM) and αKG (5 mM); viable cells were counted following trypan blue staining on days 3, 5, and 7. (F) CD34 + HPCs from 4 healthy donors were seeded at 5 × 10 5 cells/mL with and without CB-839 (1 µM); viable cells were counted following trypan blue staining on days 2, 3, 4, and 5. * P
Figure Legend Snippet: Targeting glutaminase activity inhibits AML cell proliferation. (A) OCI-AML2 and MOLM-14 shGLS1#5 leukemic cells were seeded at 5 × 10 5 cells/mL with or without doxycycline; viable cells were counted manually following trypan blue staining on days 3, 5, and 7. Each experiment was performed independently ≥3 times. (B) OCI-AML2, MV4-11, MOLM-14, HL-60, and OCI-AML3 cells transfected with shGLS1#5 or #7 were seeded at 5 × 10 5 cells/mL with or without doxycycline; viable cells were counted following trypan blue staining on days 3, 5, and 7. Each experiment was performed independently ≥3 times. (C) OCI-AML2 and MOLM-14 shGLS1#5 leukemic cells were seeded at 5 × 10 5 cells/mL with or without doxycycline, and αKG (5 mM) and viable cells were counted following trypan blue staining on day 7. (D) A total of 10 cell lines were seeded at 5 × 10 5 cells/mL with or without CB-839 (1 µM); viable cells were counted following trypan blue staining on days 3, 5, and 7. (E) OCI-AML2 cells were seeded at 5 × 10 5 cells/mL with or without CB-839 (1 µM) and αKG (5 mM); viable cells were counted following trypan blue staining on days 3, 5, and 7. (F) CD34 + HPCs from 4 healthy donors were seeded at 5 × 10 5 cells/mL with and without CB-839 (1 µM); viable cells were counted following trypan blue staining on days 2, 3, 4, and 5. * P

Techniques Used: Activity Assay, Staining, Transfection

GLS1 controls AML survival by modulating the TCA cycle. (A) ShGLS1#5 was induced with doxycycline for 4 days in OCI-AML2, HL-60, MV4-11, MOLM-14, and OCI-AML3 leukemic cells, and apoptosis was evaluated based on Annexin-V binding. (B) CD34 + HPCs from 3 healthy donors were transfected with a lentiviral vector that expressed a noninducible GLS1 shRNA (#5), and transfected cells were selected with puromycin for 2 days; 3 days later, apoptosis was evaluated based on Annexin-V staining, and protein extracts were immunobloted with anti-GAC and anti-ACTIN antibodies. (C) MOLM-14 shGLS1 cells, MOLM-14 shCTR cells (9 mice each), and OCI-AML2 shGLS1 cells or OCI-AML2 shCTR cells (9 mice each) were intravenously injected into NSG mice (2 × 10 6 cells per mouse). Mice were treated with doxycycline by oral gavage. The Kaplan-Meier survival curves of mice treated with doxycycline are shown for both cell lines. (D) The spleens of the mice injected with the OCI-AML2 shGLS1 and OCI-AML2 shCTR cell lines were measured. (E) A total of 11 cell lines were cultured for 4 days with or without CB-839 (1 µM), and apoptosis was evaluated based on Annexin-V staining. (F) OCI-AML2 and HL-60 cells were cultured for 4 days with or without CB-839 (1 µM), and dmαKG (5 mM) and apoptosis was evaluated based on Annexin-V staining. (G) OCI-AML2 cells that were stably infected with GAC WT or GAC K320A were cultured for 2 days with or without doxycycline or CB-839 (1 µM). Apoptosis was evaluated based on Annexin-V staining. (H) AML samples from 10 patients and CD34 + HPCs from 6 healthy donors were cultured for 4 days with or without CB-839 (1 µM). Apoptosis was evaluated based on Annexin-V staining. * P
Figure Legend Snippet: GLS1 controls AML survival by modulating the TCA cycle. (A) ShGLS1#5 was induced with doxycycline for 4 days in OCI-AML2, HL-60, MV4-11, MOLM-14, and OCI-AML3 leukemic cells, and apoptosis was evaluated based on Annexin-V binding. (B) CD34 + HPCs from 3 healthy donors were transfected with a lentiviral vector that expressed a noninducible GLS1 shRNA (#5), and transfected cells were selected with puromycin for 2 days; 3 days later, apoptosis was evaluated based on Annexin-V staining, and protein extracts were immunobloted with anti-GAC and anti-ACTIN antibodies. (C) MOLM-14 shGLS1 cells, MOLM-14 shCTR cells (9 mice each), and OCI-AML2 shGLS1 cells or OCI-AML2 shCTR cells (9 mice each) were intravenously injected into NSG mice (2 × 10 6 cells per mouse). Mice were treated with doxycycline by oral gavage. The Kaplan-Meier survival curves of mice treated with doxycycline are shown for both cell lines. (D) The spleens of the mice injected with the OCI-AML2 shGLS1 and OCI-AML2 shCTR cell lines were measured. (E) A total of 11 cell lines were cultured for 4 days with or without CB-839 (1 µM), and apoptosis was evaluated based on Annexin-V staining. (F) OCI-AML2 and HL-60 cells were cultured for 4 days with or without CB-839 (1 µM), and dmαKG (5 mM) and apoptosis was evaluated based on Annexin-V staining. (G) OCI-AML2 cells that were stably infected with GAC WT or GAC K320A were cultured for 2 days with or without doxycycline or CB-839 (1 µM). Apoptosis was evaluated based on Annexin-V staining. (H) AML samples from 10 patients and CD34 + HPCs from 6 healthy donors were cultured for 4 days with or without CB-839 (1 µM). Apoptosis was evaluated based on Annexin-V staining. * P

Techniques Used: Binding Assay, Transfection, Plasmid Preparation, shRNA, Staining, Mouse Assay, Injection, Cell Culture, Stable Transfection, Infection

7) Product Images from "Autocrine IGF-1/IGF-1R signaling is responsible for constitutive PI3K/Akt activation in acute myeloid leukemia: therapeutic value of neutralizing anti-IGF-1R antibody"

Article Title: Autocrine IGF-1/IGF-1R signaling is responsible for constitutive PI3K/Akt activation in acute myeloid leukemia: therapeutic value of neutralizing anti-IGF-1R antibody

Journal: Haematologica

doi: 10.3324/haematol.2009.010785

The IGF-1/IGF-1R pathway is functional and constitutively activated in PI3K + AML blast cells. (A) Expression of IGF-1R in total cell lysates from six AML samples was compared with that in normal CD34 + hematopoietic progenitors. Protein extracts from 10 6 cells were analyzed by western blotting. (B) After purification, PI3K + or (C) PI3K − AML cells were starved for 4 h in cytokine and serum-free medium and then stimulated or not with 50 ng/mL IGF-1 for 10 min. Protein extracts from 10 6 cells were analyzed by western blotting.
Figure Legend Snippet: The IGF-1/IGF-1R pathway is functional and constitutively activated in PI3K + AML blast cells. (A) Expression of IGF-1R in total cell lysates from six AML samples was compared with that in normal CD34 + hematopoietic progenitors. Protein extracts from 10 6 cells were analyzed by western blotting. (B) After purification, PI3K + or (C) PI3K − AML cells were starved for 4 h in cytokine and serum-free medium and then stimulated or not with 50 ng/mL IGF-1 for 10 min. Protein extracts from 10 6 cells were analyzed by western blotting.

Techniques Used: Functional Assay, Expressing, Western Blot, Purification

8) Product Images from "Control of Adipose Tissue Inflammation Through TRB1"

Article Title: Control of Adipose Tissue Inflammation Through TRB1

Journal: Diabetes

doi: 10.2337/db09-1537

TRB1 expression in WAT is elevated in acute and chronic inflammation. A–C : Quantitative PCR analysis of TRB1, TRB2, or TRB3 mRNA levels in abdominal WAT of wild-type (wt) and obese db/db mice ( A ), healthy control and tumor-bearing cachectic (C26) CD2F1 mice ( B ), and control (PBS)- and LPS-injected C57BL6 mice (means ± SE, n = 5). * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001. D : Western blot of WAT extracts from two representative control (PBS)- or LPS-injected animals as in C using antibodies against TRB1 or valosin-containing protein (VCP). Relative amounts of TRB1 protein levels normalized to VCP protein levels shown. AU, arbitrary units.
Figure Legend Snippet: TRB1 expression in WAT is elevated in acute and chronic inflammation. A–C : Quantitative PCR analysis of TRB1, TRB2, or TRB3 mRNA levels in abdominal WAT of wild-type (wt) and obese db/db mice ( A ), healthy control and tumor-bearing cachectic (C26) CD2F1 mice ( B ), and control (PBS)- and LPS-injected C57BL6 mice (means ± SE, n = 5). * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001. D : Western blot of WAT extracts from two representative control (PBS)- or LPS-injected animals as in C using antibodies against TRB1 or valosin-containing protein (VCP). Relative amounts of TRB1 protein levels normalized to VCP protein levels shown. AU, arbitrary units.

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Mouse Assay, Injection, Western Blot

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