Figure 3.
CXCL12 induction of resistance to venetoclax requires CD44. (A-C) OCI-AML3 cells transiently transfected with either CD44 siRNA or control siRNA were preincubated with CXCL12 and/or HA followed by venetoclax or DMSO treatment. Lysates were probed by western blot. A representative picture is shown. Data from 3 independent experiments were quantified. β-Actin was used as a loading control (Ctrl). The numbers above the C-Cas3 panel indicate fold changes. Three independent experiments were quantified (Kruskal-Wallis test). (D) OCI-AML3 cells and (E) venetoclax-resistant Molm13 (Molm13-VR) cells were transduced with CRISPR/Cas9 lentiviral vectors encoding either Scr or CD44 sgRNA, followed by puromycin selection and FACS sorting for cells not expressing CD44. Cells with or without CXCL12 induction were seeded in HA or solvent (phosphate-buffered saline)-coated tissue culture plates, followed by venetoclax or DMSO treatment. Viable cells were determined by flow cytometry using annexin V and PI staining, and data were analyzed using 1-way ANOVA. (F) Molm13-VR cells were preincubated with CXCR4 inhibitors (AMD3100 or WZ811) and induced with CXCL12, followed by venetoclax or DMSO treatment. Viable cells were determined by flow cytometry using annexin V and PI staining, and data were analyzed using the Student t test. (G) Primary AML cells were preincubated with an anti-CD44 antibody (IM7) or the corresponding isotype (immunoglobulin G [IgG]), seeded in HA-coated tissue culture plates, and induced with CXCL12 (200 ng/mL). After 3 hours, the cells were stained with annexin V and PI and analyzed by FACS. The results of each patient sample show the percentage of viable cells normalized as a ratio between control cells (incubated with IgG control and DMSO) and the viable cells in each of the other conditions (n = 4 patient samples [2 FLT3-ITD, 2 normal]) in independent experiments; data were analyzed using 1-way ANOVA. (H-I) OCI-AML3 cells transiently transfected with either CD44 siRNA or control siRNA were induced with or without CXCL12. mRNA expression of CD44, BCL-2, and BAX were determined by qPCR from 4 independent experiments; data were analyzed using a Wilcoxon rank-sum test. *P < .05; **P < .01; ***P < .001; ****P < .0001.

CXCL12 induction of resistance to venetoclax requires CD44. (A-C) OCI-AML3 cells transiently transfected with either CD44 siRNA or control siRNA were preincubated with CXCL12 and/or HA followed by venetoclax or DMSO treatment. Lysates were probed by western blot. A representative picture is shown. Data from 3 independent experiments were quantified. β-Actin was used as a loading control (Ctrl). The numbers above the C-Cas3 panel indicate fold changes. Three independent experiments were quantified (Kruskal-Wallis test). (D) OCI-AML3 cells and (E) venetoclax-resistant Molm13 (Molm13-VR) cells were transduced with CRISPR/Cas9 lentiviral vectors encoding either Scr or CD44 sgRNA, followed by puromycin selection and FACS sorting for cells not expressing CD44. Cells with or without CXCL12 induction were seeded in HA or solvent (phosphate-buffered saline)-coated tissue culture plates, followed by venetoclax or DMSO treatment. Viable cells were determined by flow cytometry using annexin V and PI staining, and data were analyzed using 1-way ANOVA. (F) Molm13-VR cells were preincubated with CXCR4 inhibitors (AMD3100 or WZ811) and induced with CXCL12, followed by venetoclax or DMSO treatment. Viable cells were determined by flow cytometry using annexin V and PI staining, and data were analyzed using the Student t test. (G) Primary AML cells were preincubated with an anti-CD44 antibody (IM7) or the corresponding isotype (immunoglobulin G [IgG]), seeded in HA-coated tissue culture plates, and induced with CXCL12 (200 ng/mL). After 3 hours, the cells were stained with annexin V and PI and analyzed by FACS. The results of each patient sample show the percentage of viable cells normalized as a ratio between control cells (incubated with IgG control and DMSO) and the viable cells in each of the other conditions (n = 4 patient samples [2 FLT3-ITD, 2 normal]) in independent experiments; data were analyzed using 1-way ANOVA. (H-I) OCI-AML3 cells transiently transfected with either CD44 siRNA or control siRNA were induced with or without CXCL12. mRNA expression of CD44, BCL-2, and BAX were determined by qPCR from 4 independent experiments; data were analyzed using a Wilcoxon rank-sum test. *P < .05; **P < .01; ***P < .001; ****P < .0001.

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