Fig 1: USP27X enhances CBX2 protein stability.A USP27X was knocked down in MCF7 and MDA-MB-231 cells by two independent shRNAs. CBX2 protein expression level was detected by IB analysis. B, C USP27X WT or C87A mutants were overexpressed in BT549 (B) and HEK-293T cells (C) The level of CBX2 protein was analyzed. D, E qRT-PCR analysis of CBX2 mRNA expression in MCF7 (D) and MDA-MB-231 (E) cell lines depleted of USP27X. F MDA-MB-231 cells transfected with 2 independent USP27X shRNAs were treated with or without the proteasome inhibitor MG132 (20 μM, 8 h) or CQ (20 μM, 12 h), and then analyzed for USP27X and CBX2. G MCF7 cells transfected with two independent USP27X shRNAs were treated with or without the proteasome inhibitor MG132 (20 μM, 8 h), and then analyzed for USP27X and CBX2. H BT549 cells transfected with USP27X WT or C87A mutants were treated with or without the proteasome inhibitor MG132 (20 μM, 8 h), and then analyzed for USP27X and CBX2. I–L MDA-MB-231 (I) and MCF7 (K) cells stably expressing control shRNA or USP27X shRNA were treated with 100 μg/ml CHX, harvested at the indicated times, and treated with IB with anti-CBX2 and USP27X antibodies. Quantification of CBX2 levels relative to Vinculin is shown. J Analyzed results of relative CBX2 level in MDA-MB-231. L Analyzed results of relative CBX2 level in MCF7. M BT549 cells transfected with USP27X WT or C87A mutants were treated with 100 μg/ml CHX, harvested at the indicated times, and treated with IB with anti-CBX2 and USP27X antibodies. Quantification of CBX2 levels relative to Vinculin is shown. Data are represented as mean ± SD of 3 independent experiments. ***P < 0.001, one-way ANOVA with Dunnett’s post test (D, E, J, L, M).