Figure 7
SiRNA-mediated knockdown of Rac1b enhances TGF-β1-mediated growth inhibition. (A) Panc1 or HPDE6c7 cells were transiently transfected twice with 50 nM each of Co siRNA or Rac1b siRNA followed by another 24-h incubation in normal growth medium. Cells were then stimulated, or not, with TGF-β1 for 24 h. In every transfection, aliquots of cells were tested for successful inhibition of siRNA targets by qPCR analysis before and after the assay (not shown). DNA synthesis in [3H]-thymidine-pulsed cells was measured during the last 4 h of the TGF-β stimulation period. Data represent the mean ± SD of six wells processed in parallel. Asterisks indicate significance relative to the respective Co siRNA-transfected sample. Data are displayed as % inhibition of [3H]-thymidine uptake after TGF-β1 treatment relative to untreated control cells set arbitrarily to 100%. The assays were performed three times and data represent the mean ± SD (n = 3). (B) Panc1 cells were transfected with Rac1b siRNA and treated with TGF-β1 as outlined under (A), and subjected to sequential immunoblotting for p21WAF1, Rac1b and HSP90 without intermittent stripping. The bands shown are all from the same blot/gel and the vertical lines indicate removal of irrelevant lanes. Three experiments were performed of which one is shown.
