Figure 5.

Development of suppression to siRNA function in CaSki-derived cells stably expressing p53 siRNA, but not in the cells stably expressing lamin A/C siRNA. (a) CaSki cells were transfected with the modified pSUPER-p53 siRNA expression vector and selected in the presence of BSD. Protein samples (lanes 3–6) were prepared from the selected stable p53 siRNA cells at each passage and analyzed by Western blot for p53 and β-tubulin. The CaSki cells with or without transient transfection of p53 siRNA duplex (5 nM) were used as transient p53 siRNA controls (lanes 1 and 2). (b) Total cell RNA prepared from vector control C-V cells (lane 1) or stable p53 siRNA cells at passages 5 (lane 2) and 15 (lane 3) was examined by Northern blot for expression of antisense (As) or sense (S) strand p53 siRNA and U6 snRNA. RNA isolated from stable E7 siRNA cells (C-2, lane 4) was examined as a control by Northern blot using E7 siRNA-specific probes as described in Figure 4b. (c) CaSki cells were stably transfected with modified pSUPER-lamin A/C siRNA expression vector (pST83) and were selected in the presence of BSD. Protein samples were prepared at each cell passage as indicated and examined by Western blot using anti-lamin A/C antibody. The same membrane was reprobed with anti-β tubulin. C-V, protein samples from CaSki cells stably transfected with an empty vector only.