Figure 2. ARID1A regulates E3-ligase RNF8-mediated Chk2 ubiquitination.

(A) Left, Western blots of ARID1A and Chk2 in ARID1A-WT (HOC8 and FUOV1) and -mutant (OAW42 and EF027) ovarian cancer cells. Right, quantitative results represent the mean ± SD from 3 independent experiments. (B) Left, Western blots of ARID1A induction by doxycycline (Dox, 2 μg/mL, 3 days) in ARID1A-null OAW42 cells. Right, quantitative results represent the mean ± SD from 3 independent experiments. (C) Left, Western blots of p-Chk2 (T68) induction by ionizing radiation (IR) (10 Gy) in ARID1A-WT (HOC8 and FUOV1) and -mutant (OAW42 and EF027) ovarian cancer cells. Right, quantitative results represent the mean ± SD from 3 independent experiments. (D) Immunoblot (IB) of U2OS cells transfected with indicated plasmid and siRNA, SFB-tagged (S-tag, Flag epitope tag, and streptavidin-binding peptide tag) Chk2 (SFB-Chk2), si-Nontarget, or siRNA targeting ARID1A along with His-ubiquitin (His-Ub) constructs; Ni–nitrilotriacetic acid (Ni-NTA), nickel bead precipitate. IB, FLAG (immunoblotting by anti-FLAG antibody). (E) Immunoblot of U2OS cells transfected with indicated plasmid and siRNA, SFB-RNF8, si-Nontarget, or siRNA targeting ARID1A along with His-Ub constructs. IB, FLAG. (F) Immunoblot of U2OS cells transfected with indicated plasmid and siRNA, SFB-RNF8, SFB-RNF8 RING domain depletion (ΔRING), si-Nontarget, or siRNA targeting ARID1A along with His-Ub constructs. IB, FLAG. (G) Immunoprecipitation (IP) of SFB-RNF8 with Myc-Chk2 in U2OS cells with si-Nontarget or siRNA targeting ARID1A. (H) Left, coimmunoprecipitation (Co-IP) of endogenous RNF8 and Chk2 in HCT116-WT and ARID1A-KO (HCT116-KO) cells. Right, quantitative analysis from normalization of Chk2 bound by RNF8 represent the mean ± SD from 3 independent experiments. One-way ANOVA with Holm-Šidák’s multiple comparisons test (A and C); 2-tailed unpaired Student’s t test (B and H). **P < 0.01; ***P < 0.001; ****P < 0.0001.