Figure 3.
PLX51107 and PLX2853 BETi-induced apoptosis is BIM dependent and effectively combines with BH3-mimetics in Eμ-myc lymphomas. (A) Eμ-myc lymphoma cell lines (n = 3) were treated as in Figure 2A and subjected to 1000 nM PLX5, 100 nM PLX2, or vehicle control (Con) for 24 hours. miR-17-5p and -92a expression was then assessed in triplicate by qPCR normalized to U6 snRNA. (B) Empty vector (GFP) or shBIM-transduced Eμ-myc lymphoma cell lines (n = 3) were treated with PLX5 or PLX2 and assessed for viability as in Figure 1A. Data represent the average of 3 independent experiments for each cell line, performed in triplicate. (Ci) Proliferation of BCL-2– (n = 4) or BCL-XL–transduced (n = 3) Eμ-myc lymphoma cell lines treated with 1000 nM PLX5, 100 nM PLX2, or a DMSO vehicle control (Con) were assessed after 48 hours and depicted as fold change in cell count compared with time 0 (T0). (Cii-iii) BCL-2–transduced (n = 3) Eμ-myc lymphoma cell lines were treated as in panel i and subjected to cell cycle analysis by hypotonic PI staining. (ii) Representative example of hypotonic PI staining. (D) BCL-2– (i) or BCL-XL–OE (ii) Eμ-myc lymphoma cell lines were treated with 1000 nM PLX5, 100 nM PLX2, or an appropriate vehicle control (Con) alongside 300 nM ABT199 (i), 10 μM WEHI-539 (WH-539) (ii), or an additional vehicle control (DMSO) for 48 hours, and viability was assessed by annexin V/PI staining. Points represent the average of 3 independent experiments, performed in triplicate, for each cell line. (E) BCL-XL–transduced Eμ-myc lymphoma cell lines (n = 3) were pretreated with 100 nM PLX2 (PLX2 PT) or a vehicle control (V PT) for 24 hours before administration of 10 μM WEHI-539 (WH-539) and 100 nM PLX2, and viability was assessed after 48 hours. Final PLX2 concentration was 100 nM in all cases. Each point represents the average of triplicate data for each cell line. Statistical analyses were performed via 2-way ANOVA. P values were adjusted for multiple comparisons using either Dunnett’s or Sidak’s analyses, where appropriate, according to experimental design. ****P < .00005, ***P < .0005, **P < .005, *P < .05. Asterisks in panel B represent overall comparisons between curves. Error bars represent SEM.

PLX51107 and PLX2853 BETi-induced apoptosis is BIM dependent and effectively combines with BH3-mimetics in Eμ-myc lymphomas. (A) Eμ-myc lymphoma cell lines (n = 3) were treated as in Figure 2A and subjected to 1000 nM PLX5, 100 nM PLX2, or vehicle control (Con) for 24 hours. miR-17-5p and -92a expression was then assessed in triplicate by qPCR normalized to U6 snRNA. (B) Empty vector (GFP) or shBIM-transduced Eμ-myc lymphoma cell lines (n = 3) were treated with PLX5 or PLX2 and assessed for viability as in Figure 1A. Data represent the average of 3 independent experiments for each cell line, performed in triplicate. (Ci) Proliferation of BCL-2– (n = 4) or BCL-XL–transduced (n = 3) Eμ-myc lymphoma cell lines treated with 1000 nM PLX5, 100 nM PLX2, or a DMSO vehicle control (Con) were assessed after 48 hours and depicted as fold change in cell count compared with time 0 (T0). (Cii-iii) BCL-2–transduced (n = 3) Eμ-myc lymphoma cell lines were treated as in panel i and subjected to cell cycle analysis by hypotonic PI staining. (ii) Representative example of hypotonic PI staining. (D) BCL-2– (i) or BCL-XL–OE (ii) Eμ-myc lymphoma cell lines were treated with 1000 nM PLX5, 100 nM PLX2, or an appropriate vehicle control (Con) alongside 300 nM ABT199 (i), 10 μM WEHI-539 (WH-539) (ii), or an additional vehicle control (DMSO) for 48 hours, and viability was assessed by annexin V/PI staining. Points represent the average of 3 independent experiments, performed in triplicate, for each cell line. (E) BCL-XL–transduced Eμ-myc lymphoma cell lines (n = 3) were pretreated with 100 nM PLX2 (PLX2 PT) or a vehicle control (V PT) for 24 hours before administration of 10 μM WEHI-539 (WH-539) and 100 nM PLX2, and viability was assessed after 48 hours. Final PLX2 concentration was 100 nM in all cases. Each point represents the average of triplicate data for each cell line. Statistical analyses were performed via 2-way ANOVA. P values were adjusted for multiple comparisons using either Dunnett’s or Sidak’s analyses, where appropriate, according to experimental design. ****P < .00005, ***P < .0005, **P < .005, *P < .05. Asterisks in panel B represent overall comparisons between curves. Error bars represent SEM.

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