Transcriptional downregulation of Bcl-2 is critical to mediate PMC-A induced apoptosis.

<p>Wild type HCT116 cells were treated with indicated concentrations of PMC-A and harvested at 24 h post-exposure, lysed and immunoblotted with anti-Bcl-2 and anti-β-actin antibodies (A). β-actin was used as loading control. Downregulation of cellular Bcl-2 was amplified by increasing PMC-A dose. HCT116 wt cells were treated with indicated PMC-A concentrations, harvested after 24 h for total RNA extraction, cDNA synthesis and qRT-PCR analysis (B). A marginal (∼5-fold) decrease in Bcl-2 transcription was evident upon PMC-A treatment (p<0,01). HCT116 wt cells were transiently transfected with expression plasmids either carrying human Bcl-2 gene or mock vector only (C). Transfected cells were treated with 50 µM PMC-A before collection and flow cytometry analysis. The results from at least 3 independent experiments were shown as means ± SD. Difference of mean values between mock and Bcl-2-transfected cells were tested using paired student’s t-test; <b>**</b>P<0.01. Analysis indicates that ectopic Bcl-2 expression renders the cells more resistant against PMC-A induced apoptosis. Bcl-2 expression statuses of mock- and Bcl-2-transfected cells prior to PMC-A treatment are displayed as immunoblotting results obtained from cells that were harvested, lysed and immunoblotted with anti-Bcl-2 antibody (C). Results from one of three independent experiments are shown in immunoblotting results.