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. 2015 Jan 28;11(1):e1004943. doi: 10.1371/journal.pgen.1004943

Figure 6. DNA2 has distinct effects on Alt-EJ and end resection, compared to FANCA and PARP.

Figure 6

(A) Stable expression of DNA2 increases Alt-EJ in cells treated with an individual siRNA targeting endogenous DNA2 (siDNA2–4). U2OS EJ2-GFP cells were transduced with an expression cassette for DNA2 with a 3xFlag immunotag and silent mutations for resistance to siDNA2–4, or transduced with EV. These cell lines were treated with siDNA2–4 or siCTRL, and transfected with expression vectors for I-SceI, or a GFP expression vector in parallel to quantify percent transfected cells. Shown are the %GFP+ frequencies from the I-SceI transfections, normalized to transfected cells. *P<0.0001 (n = 9). (B) PARPi treatment and DNA2 depletion cause an approximately additive decrease in Alt-EJ. U2OS EJ2-GFP cells were treated with siCTRL or siDNA2–4 prior to expression of I-SceI and treatment with 5 μM Olaparib (Olap) or vehicle (DMSO). Shown are %GFP+ frequencies relative to siCTRL/DMSO treated cells. *P<0.0001 (n≥6). (C) The relative mRNA abundance of FANCA and DNA2 is shown for U2OS cells treated with siCTRL, siDNA2–4, siFANCA-3, and/or siFANCA-4 (qRT-PCR analysis as in Fig. 1D, fold reduction based on 2ΔΔCt). (D) RNAi targeting of DNA2 and FANCA cause an approximately additive decrease in Alt-EJ. Shown are the GFP+ frequencies of EJ2-GFP U2OS cells treated with siDNA2–4 alone or in combination with siFANCA-3 or siFANCA-4, prior to examining Alt-EJ as in B, each normalized to siCTRL. *P<0.0001 (n≥6). (E) End resection assay. Cells were detergent extracted prior to fixation and staining with RPA and DAPI, which was analyzed for individual cells by flow cytometry. Shown are representative plots for siRNA-treated (siCTRL or siDNA2 pool) U2OS cells incubated with camptothecin (CPT) or mock treated (UN). (F) RNAi targeting of DNA2 and CtIP, but not FANCA, nor treatment with Olaparib, causes a decrease in end resection. Cells were treated with siRNA and Olaparib prior to treatment with CPT for the end resection assay. Shown is the percentage of U2OS cells with chromatin-bound (i.e. detergent-resistant) RPA staining for the given siRNA and drug treatments, as quantified using the assay shown in E. *P<0.0002 (n≥3).