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. 2020 Oct 23;6(43):eaba3058. doi: 10.1126/sciadv.aba3058

Fig. 4. RING1B interacts with EWSR1-FLI1 and affects its recruitment to chromatin.

Fig. 4

(A) Western blot showing endogenous coimmunoprecipitation of RING1B with EWSR1 in the SK-ES1 cell line. (B) Western blot showing overexpression of EWSR1-FLI1-3xFlag and RING1B levels in HeLa stably transfected cells upon induction with indicated doxycycline concentrations for 24 hours. Calnexin is used as loading control. (C) Coimmunoprecipitation of RING1B with EWSR1-FLI1-3xFlag under induction conditions (0.5 μg/ml). Inputs in (A) and (C) contain 10% of immunoprecipitated material and IgG is used as control. (D) Western blot showing RING1B and EWSR1-FLI1 in cytoplasm, soluble, and bound chromatin fractions in shCTRL#1 or shRING1B#1 SK-ES1 cells. Histone H4 is used as a control of bound chromatin, and GAPDH as a control of cytoplasmic fraction. Blot quantification of the same ordered samples is depicted below. (E) ChIP-qPCR analysis of FLI1, RING1B, and H3K27ac at EWSR1-FLI1–activated enhancers of NKX2-2, SOX2, or IGF1 genes in shCTRL#2 and shRING1B#2 A673 cells. ENC1 is used as negative control region. The values of the Y axis represent the enrichment ratio of immunoprecipitated samples relative to input. Error bars indicate SD of three independent biological experiments. Statistical significance is as follows ***P < 0.001, **P < 0.01, and *P < 0.05. (F) Aggregated plot and boxplot showing the average ChIP-seq signal of RING1B and FLI1 peaks at RING1B and EWSR1-FLI1 binding sites, respectively, in shCTRL#2 and shRING1B#2 A673 cells. (G) UCSC genome browser ChIP-seq signal tracks for EWSR1-FLI1 and RING1B in shCTRL#2 and shRING1B#2 A673 cells at SOX2 and VRK1 enhancer regions.