CSR is impaired in RNF168-deficient CH12F3-2 cells (A) Western blot analysis of control or shRNF168 CH12F3-2 cells quantifying RNF168, with β-actin as the loading control. NIH 3T3 cells treated with control siRNA and siRNF168 were used as negative and positive controls for RNF168 protein expression, respectively. (B) Six individual control and seven individual shRNF168 CH12F3-2 clones were stimulated for 2 days and then analyzed for IgA expression. Values were normalized by dividing the % IgA-positive cells in the experimental group into the % IgA-positive cells in the stimulated parental CH12F3-2 cells. CSR assays were performed on each clone in triplicate, and statistical significance was evaluated by the two-tailed t test. **P = .0023 (C) Three control and three shRNF168 CH12F3-2 clones were stimulated for 2, 3, and 4 days, after which IgA expression was analyzed. CSR assays were performed in duplicate for each clone. Statistical significance was tested by two-way ANOVA. **P = .0034.

53BP1 knockdown cells, but not RNF8/168 knockdown cells, are resistant to Nutlin-3–mediated apoptosis. (A) Percentage of control, sh53BP1, shRNF8(2), and shRNF168 CH12F3-2 cells undergoing apoptosis (i.e., surface annexin V⁺) after treatment with Nutlin-3 or DMSO for 1 day. Each assay was carried out with two different clones of each population. Statistical significance was tested by two-way ANOVA. ****P <.0001. (B) Schematic illustrating two different roles for 53BP1. AID indirectly induces a DSB by the actions of the mismatch repair (MMR) and base excision repair (BER) pathways. 53BP1 is recruited to the DNA DSB site either by direct binding (via the MRN complex or dimethylated histones) or through the γH2AX-RNF8-RNF168 amplification pathway, the latter of which leads to the accumulation and stabilization of 53BP1 at the break site. These pathways ultimately lead to end-processing and resolution of the DNA break. Independent of the RNF8/168 cascade, 53BP1 could lead to p53 stabilization or enhanced transcriptional activity in response to apoptosis signaling.

shRNA-mediated RNAi knockdown of 53BP1 in CH12F3-2 cells abrogates CSR. (A) Western blot analysis of control (Left) and sh53BP1 (Right) CH12F3-2 cells quantifying 53BP1 and Msh2 (loading control). (B) Two clones of each indicated CH12F3-2 cell population were analyzed for growth in duplicate, and statistical significance was tested by two-way ANOVA. ****P <.0001. (C) Percentage of CH12F3-2 populations undergoing apoptosis (surface annexin V⁺) with and without stimulation for 2 days. The assay was carried out with at least two individual clones for each population. (D) Clonogenic survival assays with asynchronous CH12F3-2 populations exposed to varying x-ray doses. The assay was carried out with at least two individual clones for each population. (E) Six individual control and eight sh53BP1 CH12F3-2 clones were stimulated for 2 days and then analyzed for IgA expression. Values were normalized by dividing the % IgA-positive cells in the experimental group into the % IgA-positive cells in the stimulated parental CH12F3-2 cells. CSR assays were performed on each clone in duplicate, and statistical significance was tested using the one-tailed t test. (F) Three control and three sh53BP1 CH12F3-2 clones were stimulated for 2, 3, and 4 days, after which IgA expression was analyzed. CSR assays were performed in duplicate for each clone. Statistical significance tested by two-way ANOVA. ****P <.0001.

CSR is impaired in RNF8-deficient CH12F3-2 cells. (A) Western blot analysis quantifying RNF8, 53BP1, and PCNA (loading control) in CH12F3-2 cells with control shRNA and two different lentiviral vectors targeting RNF8(1) and (2). (B) Western blot analysis of stimulated and unstimulated control or shRNF8-treated CH12F3-2 cells, quantifying RNF8 and γH2AX protein levels, with PCNA as the loading control. (C) Eight individual clones for each of control, shRNF8(1), shRNF8(2), and CH12F3-2 cells were stimulated for 3 days and then analyzed for IgA expression. Values were normalized by dividing the % IgA-positive cells in the experimental group to the % IgA-positive cells in the stimulated parental CH12F3-2 cells. Statistical significance was evaluated by the two-tailed t test. *P <.05; ****P <.0001. (D) Two control, two shRNF8(1)-treated, and two shRNF8(2)-treated CH12F3-2 clones were stimulated, and IgA expression was analyzed at 2, 3, and 4 days poststimulation. Statistical significance was tested by two-way ANOVA. ***P <.001; ****P <.0001. (E) Stimulated control and shRNF8 CH12F3-2 cells were collected at times 0 h and 48 h and then stained with antibodies specific to γH2AX and 53BP1. Cells from two separate experiments (each with n = 1,000), having single γH2AX foci with 53BP1 (γH2AX ± 53BP1⁺ foci) or without 53BP1 (γH2AX ± 53BP1^- foci) colocalization were counted and analyzed as shown. More than 90% of the cells did not exhibit γH2AX foci after cytokine stimulation. Statistical significance was tested using the two-tailed t test between control 48 h and shRNF8(2) 48 h. *P = .014.