Figure 8.
Figure 8. BIM contributes to CDK inhibition-induced cell death in cells with elevated MYC expression. (A) RPE cells, with or without constitutive MYC overexpression, were treated with 10 µM purvalanol A for 72 h and were tested for protein expression of multiple pro- and antiapoptotic BCL2 family members, as well as cleaved PARP and loading control β-actin. The results shown are representatives of at least five independent experiments. (B) Relative fold change in BIM protein expression in RPE-MYC cells treated with purvalanol A for 72 h. The purvalanol A time course experiments were repeated at least five times and BIM protein was quantified as described in Materials and methods. The error bars represent means ± SEM. (C) BIM mRNA expression after purvalanol A treatment. BIM mRNA levels were quantified by qPCR in RPE cells with or without constitutive MYC overexpression. Mean of three experiments ± SEM are shown. (D) BIM expression in RPE-MYC cells treated with dinaciclib for 36 h. (E) RPE-MYC cells were transfected with either a pool of specific BIM siRNAs or a pool of control nontargeting siRNAs and cell viability was determined after treatment with 10 µM purvalanol A for 72 h. The experiment was repeated three times. The error bars represent ± SEM. (F) BIM protein expression in a panel of triple-negative cells. Cell lines were tested for BIM protein expression after treatment with 10 µM purvalanol A for 72 h. The results shown are the representatives of at least three independent experiments. (G) Quantification of BIM protein expression. Shown are the means from at least three independent experiments that yielded the following SEMs, respectively: ±0.2 (HCC3153), ±0.88 (BT549), ±0.48 (MCF10AMYC), and ±0.57 (SUM149PT). (H) shRNA-mediated BIM knockdown in two triple-negative cell lines, MCF10AMYC and BT549. shRNA against GFP was used as negative control. (I) BIM knockdown effects on cell death of MCF10AMYC and BT549 cells after purvalanol A treatment. The experiments were repeated at least three times in triplicate. Error bars represent means ± SEM. P-values were calculated by two-tailed Student’s t test.

BIM contributes to CDK inhibition-induced cell death in cells with elevated MYC expression. (A) RPE cells, with or without constitutive MYC overexpression, were treated with 10 µM purvalanol A for 72 h and were tested for protein expression of multiple pro- and antiapoptotic BCL2 family members, as well as cleaved PARP and loading control β-actin. The results shown are representatives of at least five independent experiments. (B) Relative fold change in BIM protein expression in RPE-MYC cells treated with purvalanol A for 72 h. The purvalanol A time course experiments were repeated at least five times and BIM protein was quantified as described in Materials and methods. The error bars represent means ± SEM. (C) BIM mRNA expression after purvalanol A treatment. BIM mRNA levels were quantified by qPCR in RPE cells with or without constitutive MYC overexpression. Mean of three experiments ± SEM are shown. (D) BIM expression in RPE-MYC cells treated with dinaciclib for 36 h. (E) RPE-MYC cells were transfected with either a pool of specific BIM siRNAs or a pool of control nontargeting siRNAs and cell viability was determined after treatment with 10 µM purvalanol A for 72 h. The experiment was repeated three times. The error bars represent ± SEM. (F) BIM protein expression in a panel of triple-negative cells. Cell lines were tested for BIM protein expression after treatment with 10 µM purvalanol A for 72 h. The results shown are the representatives of at least three independent experiments. (G) Quantification of BIM protein expression. Shown are the means from at least three independent experiments that yielded the following SEMs, respectively: ±0.2 (HCC3153), ±0.88 (BT549), ±0.48 (MCF10AMYC), and ±0.57 (SUM149PT). (H) shRNA-mediated BIM knockdown in two triple-negative cell lines, MCF10AMYC and BT549. shRNA against GFP was used as negative control. (I) BIM knockdown effects on cell death of MCF10AMYC and BT549 cells after purvalanol A treatment. The experiments were repeated at least three times in triplicate. Error bars represent means ± SEM. P-values were calculated by two-tailed Student’s t test.

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