SHLD2/FAM35A co-operates with REV7 to coordinate DNA double-strand break pathway choice. (2018)
Figure 4

Figure 4. SHLD2 is an effector of REV7 in promoting NHEJ and antagonizing HR.

(A) U2OS mCherry-LacR-Fok1 cells were treated with the indicated siRNA and subsequently transfected with a GFP-SHLD2 construct. 24 h post-transfection DNA damage was induced using Shield-1 and 4-OHT. The cells were then fixed and analyzed for the intensity of the GFP-SHLD2 signal at mCherry-LacR-Fok1 focus. Shown is the quantification of the GFP-SHLD2 signal at the Fok1 focus. Data are represented as a box-and-whisker plot where the whiskers represent the 10-90 percentile. At least 75 cells were counted per condition. Significance was determined by one-way ANOVA followed by a Dunnett test. *p<0.05.

(B) 293T cells were transfected with Flag-REV7 and GFP-SHLD2 expression vectors as indicated. 24h post-transfection cells were treated with DMSO or with 10uM of ATM inhibitor KU-60019 for 1 h prior to irradiation. 1h post-irradiation (10 Gy) nuclear extracts were prepared and REV7 complexes were immunoprecipitated using anti-Flag (M2) Resin and then analyzed by immunoblotting using GFP, REV7 and p-Chk1 antibodies.

(C) U2OS EJ5-GFP cells were transfected with the indicated siRNAs. At 24hr post-transfection, cells were transfected with the I-SceI expression plasmid, and the GFP+ population was analyzed 48 hr post-plasmid transfection. The percentage of GFP+ cells was determined for each individual condition and subsequently normalized to the non-targeting condition (siCTRL). Data are presented as the mean ± SD (n=3). Significance was determined by one-way ANOVA followed by a Dunnett test using Ctrl+SceI as a comparison (*p<0.0005) or the indicated reference (n.s.= non-significant).

(D) CH12F3-2 cells stably expressing the indicated shRNAs were stimulated with a cocktail of cytokines (CIT) to induce class switching to IgA. The percentage of IgA+ cells was monitored 24 and 48h post-stimulation by staining with an anti-IgA antibody followed by flow cytometry analysis. Data are presented as the mean ± SD (n=3). Significance was determined by one-way ANOVA followed by a Dunnett test. *p<0.05, **p<0.005, ***p<0.0005.

(E) U2OS DR-GFP cells were transfected with the indicated siRNAs. At 24hr post-transfection, cells were transfected with the I-SceI expression plasmid, and the GFP+ population was analyzed 48 hr post-plasmid transfection. The percentage of GFP+ cells was determined for each individual condition and subsequently normalized to the non-targeting condition (siCTRL). Data are presented as the mean ± SD (n=3). Significance was determined by one-way ANOVA followed by a Dunnett test. *p<0.005, **p<0.0005.

(F) Schematic representation of the DNA fiber assay experimental design (Left). U2OS cells were transfected with the indicated siRNAs and then treated with CldU, IdU and NCS 48h post-transfection as indicated. The slides were stained, dehydrated, mounted and visualized and shown is the quantification of CldU/IdU tract length in order to visualize DNA end-resection (Right). At least 500 DNA tracks were measured per condition. Data are represented as a box-and-whisker plot where the whiskers represent the 10-90 percentile. Significance was determined by one-way ANOVA followed by a Dunnett test. *p<0.0005.