Figure 6.

CtIP-dependent miRNAs are not essential for HR.A, diagram showing the function of the EGFP-HR DSB repair reporter. B, schematic of HR assays workflow. U2OS cells carrying EGFP-HR reporter were transfected with synthesized miRNA mimics; DSB was induced by I-SceI–containing lentivirus, and EGFP-positive signals were analyzed by flow cytometry (fluorescence-activated cell sorting). C, scatter plot showing the relative levels of EGFP-HR reporter after all individual miRNAs mimicked transfection. HR value in miRNA control mimic-transfected cells was set as 1 for normalization. The dotted line represents the cutoff used to determine decreased and increased HR. MiRNAs that suppressed HR efficiency are indicated at the top. D–F, end resection function of CtIP is DGCR8 independent. D and E, U2OS cells were transfected with indicated siRNA or siRNA combination, treated with CPT (2 μM) for 1 h followed by RPA phosphorylation analysis by Western blotting (D) and RPA foci formation analysis by immunostaining (E) with indicated antibodies. E, the pink and white arrows indicate representative RPA2 and γH2AX foci-positive and foci-negative cells, respectively. The scale bars represent 10 μm. The percentage of RPA2 foci–positive cells among γH2AX-positive cells for each sample is shown. Data shown represent the means of three independent experiments, with error bars as SD. The p value is indicated as ∗∗p < 0.01. F, schematic and quantification of a quantitative PCR–based cellular resection assay of ER-AsiSI U2OS cells transfected with indicated siRNA or siRNA combination. Data represent the means ± SD of three independent experiments. The p value is indicated as ∗∗p < 0.01. CtIP, C-terminal-binding protein–interacting protein; DGCR8, DiGeorge syndrome critical region gene 8; DSB, DNA double-strand break; EGFP, enhanced GFP; HR, homologous recombination; n.s., not significant; RPA, replication protein A; U2OS, human osteosarcoma cell line.