Reduction of CENPA overexpression by RNAi in pRb depleted cells. A) Western blot showing CENP-A and CENP-F protein levels in HCT116 cells, wild type (HCT, lane 1), p53-knockout (HCTp53KO, lane 3) and after pRb acute loss (siRB+, lanes 2 and 4), β-tubulin was used as a loading control. B) Real-time RT-PCR showed CENPA decreased transcript levels after simultaneous RB/CENPA post-transcriptional silencing (siRB/CenpA), in comparison to CENPA expression in RB-depleted cells (siRB). RB transcripts were reduced in both pRb- and pRb/CenpA- depleted cells. Untransfected HCT116 cells were used as a calibrator. C) Western blot showing reduction of CENP-A protein levels, in comparison to CENPA expression in RB-depleted cells (siRB+), in cells simultaneously transfected with siRNAs specific for both RB and CENPA (lane 3). Protein extracts of HCT116 wild type and pRb-depleted cells were loaded in lanes 1 and 2 respectively. β-tubulin was used as a loading control. D) Real-time RT-PCR showing CENPA transcript levels in HCT116 cells transfected with a control siRNA targeting GFP (siGFP) and after transfection of siRNAs targeting CENPA (two different doses siCenpA 60 nM, siCenpA 80 nM). E) Western blot showing reduction of CENP-A protein levels after CenpA post-transcriptional silencing (lane 3: 60 nM, lane 4: 80 nM). Normal levels of CENPA were present in untransfected cells (lane 1) and in cells transfected with a control siRNA (lane 2: siGFP).