Figure 9.

Role of IBRDC2 in the regulation of apoptosis. (A) IBRDC2 RNAi and Control RNAi cells were treated with 1 μM STS or 10 μM ActD for 3 h and 6 h and analysed with western blot for PARP cleavage, caspase-3 and caspase-9 activation, IBRDC2 and GAPDH (loading control). ‘N' refers to full-length proteins, ‘P' processed proteins. (B) Quantification of PARP cleavage in IBRDC2 RNAi versus Control RNAi cells. Western blots were scanned and intensities of cleaved PARP bends were quantified using ImageJ software. The data were normalized with cleaved PARP in Control RNAi taken as 1.0 in each analysed sample (n=3). (C) Untreated IBRDC2 RNAi (blue lines; two bottom panels) and Control RNAi (blue lines; two top panels), and cells treated with ActD (black lines) and STS (black lines) for 5 h were incubated for 1 h with caspase-3- and caspase-7-specific FLICA reagent and then analysed with FACS. Red line indicates the border between FLICA-negative and FLICA-positive cells. (D) IBRDC2 RNAi cells and Control RNAi cells were treated as in A and analysed with western blot for changes in protein levels of Mcl-1 and p53. (E) Control vector transfected WT and Bax^−/− HCT116 cells were treated with 10 μM ActD or 1 μM STS for 10 h, followed by western blot with antibodies indicated in the figure. (F) IBRDC2 RNAi WT or Bax^−/− HCT116 cells and Control RNAi WT or Bax^−/− HCT116 cells were treated with 1 μM STS or 10 μM ActD for 3 h and 6 h and analysed for PARP processing. To confirm the RNAi efficiency in HCT116 cells, the same membranes were also stained with anti-IBRDC2 mAb, anti-Bax mAb and anti-Tom20 polyclonal antibody (loading control).