Figure 4.

Development of suppression to siRNA function in CaSki-derived cells with stable expression of E7 siRNA. (a) Reappearance of 16E7 in CaSki-derived C-2 cells stably expressing HPV16 E7 siRNA as an indication of loss of E7 siRNA function with additional cell passages. E7 expression in protein samples prepared at passage 4 and at passage 6 was compared by Western blot. (b) Loss of E7 siRNA function in the C-2 cells at later cell passages was not due to decreased expression of E7 siRNA. Total cell RNA was probed for sense (S) or antisense (As) strand siRNA expression by Northern blot with a ³²P-labeled As or S oligo or a U6-specific oligo as a normalization of sampling. (c) Expression of E7 siRNA in single-cell subclones. Total cell RNA extracted from each single-cell subclone or their parent C-2 cells (lane 9) or vector-control C-V cells (lane 8) was examined for antisense (As) strand siRNA expression by Northern blot using a ³²P-labeled sense oligo or a U6-specific oligo for sampling control. Total cell RNA isolated from CaSki cells (C, lanes 10–11) transiently transfected with NS (lane 10) or E7 siRNA (lane 11) at 10 nM was included as a cell control and a siRNA size control, respectively. (d) Expression of E7, p53, and p21 in each single-cell subclone was analyzed by Western blot with antibodies against HPV16 E7, p53, or p21. (e) Cell passage affects E7 siRNA function in single-cell subclone D4SC cells. Protein samples prepared from cell passage 4, 6, and 8 were examined by Western blot using antibodies against HPV16 E7 or cyclin A and compared with the protein levels in CaSki-derived vector control C-V cells. The membranes in panels a, d, and e were also blotted for β-tubulin as a sample loading control.