Figure 1
RNF31 depletion inhibits cell proliferation and increases G1 arrest in MCF-7 cells. (a) MCF-7 cells were transfected with siRNF31 or siControl and knockdown efficacy was determined by western blot analysis for RNF31, using GAPDH as internal standard for control (Left panel), and qPCR (Right panel). (b) The WST-1 assay was used to determine the cellular metabolic activity at indicated time points after transfection. Cells are treated for indicated times with 10 nM E2 or vehicle. Experiments were done in triplicates. *P<0.05; **P<0.01 for siRNF31 E2 versus siControl E2. (c) RNF31 knockdown induces arrest in the G1 phase and inhibits the E2-mediated progression into the S phase. The effects of RNF31 knockdown were compared with the effects of ERα knockdown in MCF-7 cells. Cells are treated for 24 h with 10 nM E2 or vehicle. The proportion of cells in each phase was measured by fluorescent-activated cell sorting (FACS). Experiments were done in triplicates. *P<0.05; **P<0.01; ***P<0.001 for siRNF31 versus siControl. All values are mean±s.d. (n=3). (d) Overexpression of ERα partially reverses the reduced number of cells in the S phase following RNF31 depletion. MCF-7 cells were cultured in 10% fetal bovine serum (FBS). The proportion of cells in each phase was measured by FACS. Experiments were done in triplicates. *P<0.05; **P<0.01 for siRNF31 versus siControl; siRNF31 versus siRNF31 plus ERα overexpression. All values are mean±s.d. (n=3).
