Figure 4
Silencing of IP 3 R3 by RNA interference limits the proliferative effect of E 2 . (A) Validation of the efficiency of the siRNAs used at the mRNA level (a, representative illustrations for 3 independent experiments). The IP3R3 mRNA level was lowered by about 70% (see text for details) at 24, 48 and 72 h post-transfection. (B) siR3 was responsible for a rapid and long-lasting diminution of the expression of IP3R3 at the protein level (a). Compared to control conditions, siR3 diminished the expression of IP3R3 at 24, 48 and 72 h (b). (C) Following IP3R3 silencing, the levels of IP3R1 and IP3R2 mRNA (a) and protein (b) were not significantly changed at 24, 48 and 72 h post-transfection. Representative blots for 3 to 4 independent western blotting experiments are shown. (D) MCF-7 cells were transfected with siC or siR3 and seeded for 18 h in 5-FCS and then starved for 6 h in 0-FCS. Cells were subsequently cultured in 0-FCS in the absence (-E2) of the presence (+E2) of E2 (10 nM) for 48 h. MCF-7 cell number was measured at 72 h post-transfection using the trypan-blue exclusion method. E2-induced increase in cell number was strongly diminished in siR3-transfected MCF-7 cells compared to siC-transfected cells (P < 0.01).