A and B, Inhibition of UBE3C suppresses estrogen-induced cell proliferation. MCF-7 cells transfected with control or UBE3C-specific siRNA (A), mock transfected, or transfected with wild-type (WT) FLAG-UBE3C or its C1051A (CA) mutant (B) were cultured for the indicated time with or without 17β-estradiol. Live cells were counted, and the mean ± SD from triplicate experiments is shown. C and D, Inhibition of UBE3C does not affect estrogen-induced transactivation by ERα. MCF-7 cells were transfected with control, UBE3C-, or ERα-specific siRNA (C), mock transfected, or transfected with WT FLAG-UBE3C or its C1051A (CA) mutant (D). Cells were additionally transfected with pGL3-3xERE-TATA-Luc and cultured for 24 hours with or without 17β-estradiol. Luciferase-based transcriptional activity values are expressed as the mean ± SD. E, Estrogen-regulated gene expression is not affected by the inhibition of UBE3C. MCF-7 cells transfected with the indicated siRNA were cultured for 6 hours with or without 17β-estradiol, and CCND1 (left panel) or FOS (right panel) mRNA expression levels were analyzed by real-time quantitative PCR. Relative ratios normalized to GAPDH mRNA expression are shown. The mean ± SD is shown.