Figure 6
Figure 6. Identification of BCL2A1 and BCL-XL as resistance factors for ABT-737. (A) Jurkat T cells were transiently transfected with pYFP-BCL2A1 (solid line) or empty vector control (dotted line) using Amaxa program C-16. After transfection for 16 hours, cells were exposed to different ABT-737 concentrations for 4 hours and apoptosis was assessed by PS externalization in the YFP-positive population. Data are mean plus SEM of 5 independent experiments. Statistics were done using t test (*P < .05). Inset shows expression of BCL2A1 in the total population by Western blotting in one representative experiment. (B-D) CLL cells were transfected with siRNA against BCL-X, BCL2A1, or negative control siRNA using Amaxa program X-5. Directly after nucleofection, cells were cultured on CD154-expressing cells in the presence of IL-4 (10 ng/mL) for 16 hours. (B) Knockdown of BCL-XL and BCL2A1 was confirmed by Western blotting. (C) After knockdown of BCL-XL or BCL2A1, CLL cells were removed from CD154-expressing cells and exposed to different concentrations of ABT-737 for 4 hours. Apoptosis was assessed by PS externalization and AnnexinV/FITC binding (n = 15). Statistics were done using one-way ANOVA followed by Bonferroni multiple comparison test. The BCL2A1 siRNA showed significant difference to both CD154 untransfected as well as CD154 negative siRNA group (*P < .01), while BCL-X siRNA showed significant difference only compared with CD154 untransfected group (*P < .01). (D) After combined knockdown of BCL-XL and BCL2A1, CLL cells were removed from CD154-expressing cells and exposed to different concentrations of ABT-737 for 4 hours. Apoptosis was assessed by PS externalization and annexin V/FITC binding (n = 3). In cells from another 8 patients, the combined knockdown was inefficient compared with the single knockdowns (Figure S2). Statistics were done using one-way ANOVA followed by Dunnett multiple comparison test using parental untransfected as control group. Reversal of resistance as indicated by no significant difference to control group is indicated with † (P > .05).

Identification of BCL2A1 and BCL-XL as resistance factors for ABT-737. (A) Jurkat T cells were transiently transfected with pYFP-BCL2A1 (solid line) or empty vector control (dotted line) using Amaxa program C-16. After transfection for 16 hours, cells were exposed to different ABT-737 concentrations for 4 hours and apoptosis was assessed by PS externalization in the YFP-positive population. Data are mean plus SEM of 5 independent experiments. Statistics were done using t test (*P < .05). Inset shows expression of BCL2A1 in the total population by Western blotting in one representative experiment. (B-D) CLL cells were transfected with siRNA against BCL-X, BCL2A1, or negative control siRNA using Amaxa program X-5. Directly after nucleofection, cells were cultured on CD154-expressing cells in the presence of IL-4 (10 ng/mL) for 16 hours. (B) Knockdown of BCL-XL and BCL2A1 was confirmed by Western blotting. (C) After knockdown of BCL-XL or BCL2A1, CLL cells were removed from CD154-expressing cells and exposed to different concentrations of ABT-737 for 4 hours. Apoptosis was assessed by PS externalization and AnnexinV/FITC binding (n = 15). Statistics were done using one-way ANOVA followed by Bonferroni multiple comparison test. The BCL2A1 siRNA showed significant difference to both CD154 untransfected as well as CD154 negative siRNA group (*P < .01), while BCL-X siRNA showed significant difference only compared with CD154 untransfected group (*P < .01). (D) After combined knockdown of BCL-XL and BCL2A1, CLL cells were removed from CD154-expressing cells and exposed to different concentrations of ABT-737 for 4 hours. Apoptosis was assessed by PS externalization and annexin V/FITC binding (n = 3). In cells from another 8 patients, the combined knockdown was inefficient compared with the single knockdowns (Figure S2). Statistics were done using one-way ANOVA followed by Dunnett multiple comparison test using parental untransfected as control group. Reversal of resistance as indicated by no significant difference to control group is indicated with † (P > .05).

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