Figure 2.
CXCL12 and HA protect AML cells from venetoclax-induced apoptosis. (A) OCI-AML3 cells were preincubated with CXCL12 and/or HA where indicated, followed by venetoclax or solvent (DMSO) treatment. Lysates were probed with the antibodies indicated in the western blot. Data from 1 representative of 3 experiments are shown. The numbers above the C-Cas3 panel indicate fold changes. β-Actin was used as a loading control. Three independent experiments were quantified (Student t test). (B) 2 × 106 primary cells per mL from AML patient samples were seeded into HA-coated wells or control wells for 30 minutes in technical triplicates and incubated with CXCL12 200 ng/mL. After 1 hour, venetoclax 1 µM or DMSO (vehicle) was added to the corresponding wells. After 3 hours of incubation, the cells were harvested and stained with annexin V-fluorescein isothiocyanate (FITC) and PI and analyzed by flow cytometry. The frozen patient samples contained a population of PI+ cells, which could not be distinguished from PI+ cells at the end of the experiment, so the results were calculated using the percentage of viable cells (annexin V–/PI–). The value of each sample was expressed as a ratio between the percentage of viable control cells (incubated in the absence of HA and/or CXCL12 and treated with DMSO) and the percentage of viable cells in each of the other conditions of the same patient sample to normalize for basal differences between samples of different patients (n = 5 patient samples [3 FLT3-internal tandem duplication [FLT3-ITD]; 2 normal]) in independent experiments (1-way ANOVA). (C) OCI-AML3 cells with or without CXCL12 induction were seeded in HA-coated tissue culture plates and treated with venetoclax. Apoptotic cells (annexin V+) of the adherent and suspended populations were determined separately by flow cytometry. Three independent experiments were quantified (1-way ANOVA). (D) Primary AML cells were incubated with CXCL12 and/or HA coating for 4 hours. mRNA expression of BCL-2 and MCL-1 were determined by qPCR from 3 independent experiments with 3 different primary AML samples (Kruskal-Wallis test). *P < .05; **P < .01; ****P < .0001. The error bars indicate standard deviation. ns, not significant.

CXCL12 and HA protect AML cells from venetoclax-induced apoptosis. (A) OCI-AML3 cells were preincubated with CXCL12 and/or HA where indicated, followed by venetoclax or solvent (DMSO) treatment. Lysates were probed with the antibodies indicated in the western blot. Data from 1 representative of 3 experiments are shown. The numbers above the C-Cas3 panel indicate fold changes. β-Actin was used as a loading control. Three independent experiments were quantified (Student t test). (B) 2 × 106 primary cells per mL from AML patient samples were seeded into HA-coated wells or control wells for 30 minutes in technical triplicates and incubated with CXCL12 200 ng/mL. After 1 hour, venetoclax 1 µM or DMSO (vehicle) was added to the corresponding wells. After 3 hours of incubation, the cells were harvested and stained with annexin V-fluorescein isothiocyanate (FITC) and PI and analyzed by flow cytometry. The frozen patient samples contained a population of PI+ cells, which could not be distinguished from PI+ cells at the end of the experiment, so the results were calculated using the percentage of viable cells (annexin V–/PI–). The value of each sample was expressed as a ratio between the percentage of viable control cells (incubated in the absence of HA and/or CXCL12 and treated with DMSO) and the percentage of viable cells in each of the other conditions of the same patient sample to normalize for basal differences between samples of different patients (n = 5 patient samples [3 FLT3-internal tandem duplication [FLT3-ITD]; 2 normal]) in independent experiments (1-way ANOVA). (C) OCI-AML3 cells with or without CXCL12 induction were seeded in HA-coated tissue culture plates and treated with venetoclax. Apoptotic cells (annexin V+) of the adherent and suspended populations were determined separately by flow cytometry. Three independent experiments were quantified (1-way ANOVA). (D) Primary AML cells were incubated with CXCL12 and/or HA coating for 4 hours. mRNA expression of BCL-2 and MCL-1 were determined by qPCR from 3 independent experiments with 3 different primary AML samples (Kruskal-Wallis test). *P < .05; **P < .01; ****P < .0001. The error bars indicate standard deviation. ns, not significant.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal