Figure 4

pRb acute loss induced differential expression of several centrosome and mitotic genes. A) Real-time RT-PCR showed increased expression levels of genes involved in centrosome duplication (PLK1, AURKA, and CYCE), as well in the SAC and mitosis (BRCA1, PTTG1, CDC20, BUBR1, MAD2, CENPA, and CENPF) after pRb acute loss in both HCT116wt (HCT) and p53-knockout (HCTp53KO) cells. The x-axis indicates the genes and the y-axis the relative quantification in pRb-depleted cells in respect to the gene expression level in control cells, (untransfected HCT116wt and HCT116p53KO were used as calibrator). B) Western blot showing Mad2, AurkA, Plk1, p53 and BRCA1 protein levels in HCT116 cells both wild-type (HCT, lane 1) and p53-knockout (HCTp53KO, lane 3) and after pRb acute loss (siRB+, lane 2 and lane 4 respectively). β-tubulin was used as a loading control. C) Real-time RT-PCR showing that changes in MAD2 transcript depended on BRCA1 gene expression levels. BRCA1 transcripts were reduced more than 80% at 72 hours post-transfection of siRNAs targeting BRCA1 (siBrca1 60 nM). Modulation of BRCA1 transcript levels in RB-depleted cells using different siRNA concentration (50 nM and 60 nM) reduced MAD2 expression accordingly. D) Graph showing mitotic indices in both wild type and p53-knockout HCT116 cells after pRb acute loss (siRB) and in released cells in comparison with untransfected cells (untr). E) Western blot showing similar BubR1 protein levels in untransfected cells (lanes 1 and 4) and in RB depleted cells (lanes 3 and 6). As expected HCT116 and HCTp53KO cell types after colcemid treatment (lanes 2 and 5) showed increase in BubR1 protein levels.