Figure 3

The control of the increased cdc42 protein expression does not occur at the level of transcription or altered protein stability. Analysis of differentially expressed mRNAs after increased eIF6 expression was performed on different target genes in A2780 ovarian cancer cells. A) qPCR of cdc42 mRNA was performed analysing 2 μg of total RNA reverse-transcribed into cDNA and comparing its expression between A2780 ovarian cancer cells over-expressing eIF6 with respect the control. The bar graphs represent the relative fold changes of cdc42 mRNA presented as mean ± S.D. and relative to that of GAPDH. The results are the average of three independent experiments. B) qPCR of synd-1, hax1 and hgf mRNA was performed analysing 1μg of total RNA reverse-transcribed into cDNA and comparing its expression between A2780 ovarian cancer cells over-expressing eIF6 with respect the control. The bar graphs represent the relative fold changes of target mRNAs presented as mean ± S.D. and relative to that of GAPDH. The results are the average of three independent experiments. The statistical analysis was performed with the t-test and the P-values were < 0.02 (**) and < 0.001 (*), respectively. C-D) To examine the stability of cdc42 protein, A2780 cells over-expressing eIF6 and the corresponding control were treated 24 hours after their transfection with 15 μM of the protein synthesis inhibitor CHX for the next 15 hours. Successively, endogenous cdc42 protein expression was detected by western blot analysis with an anti-cdc42 antibody and the intensity of the bands was normalized with respect the endogenous levels of β-tubulin. The expression levels of Cdc42 were determined by densitometry using ImageJ software. Results are shown for two of three independent experiments and are presented as mean ± S.D.