Fig 1: PHF6 controls classical NHEJ A, BU2OS cells expressing GFP-PHF6 and mCherry-NBS1 were locally irradiated in the absence and presence of ATM and ATR inhibitors. Shown is the recruitment of GFP-PHF6 (A) or mCherry-NBS1 (B) to laser-induced breaks in time. At least 30 cells were analyzed in three individual experiments, and error bars represent the SD.CU2OS-DR cells containing the I-SceI HR reporter were transfected with the indicated siRNA oligos and I-SceI. 48 h later, the cells were fixed and analyzed by flow cytometry. Represented is the relative repair efficiency as compared to the Luciferase control. Error bars represent the SEM of three independent experiments. Statistical significance was determined using a two-tailed, unpaired t-test (**P < 0.01, ***P < 0.001, ****P < 0.0001).DU2OS cells depleted of PHF6 with three different siRNA oligonucleotides and U2OS WT and PHF6 knockout cells were lysed and analyzed using Western blotting with the indicated antibodies.EU2OS WT and PHF6 knockout cells transfected with siRNA oligos against luciferase or XRCC4 were lysed and analyzed using Western blotting with the indicated antibodies. The remaining PHF6 signal in the PHF6 KO sample is likely due to a cross reaction at similar height as PHF6 (also see Fig EV3C).F, GDistribution of the deletion sizes (F) or the extent of microhomology for the category simple deletions (G) obtained from independently obtained I-SceI repair events (described in Fig 4E) in the indicated samples.
Fig 2: PHF6 affects recovery by controlling DNA repair via classical NHEJ A, BU2OS cells stably expressing GFP-PHF6 and mCherry-NBS1 were laser-irradiated, and cells were analyzed by time-lapse imaging. In (B), the PARP inhibitor Olaparib was added 30 min before laser-induced damage. At least 60 cells were analyzed in three individual experiments, and error bars represent the SD (lower panel). Scale bar is 5 µm.CGC92 SV40 immortalized human fibroblasts containing the I-SceI NHEJ reporter were transfected with the indicated siRNA oligos and I-SceI. 48 h later, the cells were fixed and analyzed by flow cytometry. Represented is the relative repair efficiency as compared to the Luciferase control. Error bars represent the SEM of three independent experiments. Statistical significance was determined using a two-tailed, unpaired t-test (**P < 0.01, ***P < 0.001).DClonogenic survival assays of U2OS WT and PHF6 knockout cells that were depleted for luciferase or XRCC4 and treated with 2, 3, or 4 Gy IR. Shown is the relative survival as compared to the undamaged control. Error bars represent the SEM of three individual experiments.EGC92 SV40 immortalized human fibroblasts containing the I-SceI NHEJ reporter were transfected with the indicated siRNA oligos and I-SceI. 48 h later, genomic DNA was extracted and repair of I-SceI cut sites analyzed through junction analysis (n = independently derived sequences, see Materials and Methods for details).FModel showing early recruitment of PHF6 to DNA DSBs in a PARP1/2-dependent manner, possibly to remodel the chromatin for efficient DNA repair through classical NHEJ and thereby to promote recovery from the G2 checkpoint arrest.
Fig 3: Depletion of PHF2 impairs DSB repair by homologous recombination. (A) U2OS cells, transfected with Luc or PHF2 siRNA oligos, and 24 h later transfected with GFP-CtIP and mCherry-Nbs1, were laser-irradiated and analysed by time-lapse imaging. Upper panel: representative images of the indicated time points. Lower panel: relative fluorescence (left: GFP-CtIP, right: mCherry-Nbs1) at laser stripes of at least 50 cells per experiment. (B) U2OS cells were depleted for Luc or PHF2 by siRNA, treated with IR (10 Gy) and subjected to chromatin fractionation (WCE: whole cell extracts, Sol: soluble and Chrom: chromatin). Samples were analysed by western blot using the indicated antibodies. (C) U2OS cells were depleted for Luc, CtIP or PHF2 by siRNA. Cells were treated with IR (3 Gy), fixed after 4 h and Rad51 focus formation was analysed by immunofluorescence. Top panel: representative images. Bottom panel: quantification from three independent experiments with each at least 100 cells. (D) U2OS cells stably expressing a single copy of the DR-GFP reporter construct were depleted of CtIP, PHF2 or control (non-target, NT). After 48 h, GFP fluorescence was analysed by flow cytometry. Presented is the relative fluorescence as compared to the control cells, of three independent experiments.
Fig 4: Characterization of PHF3, ACTL6A, and BRD2 in G2 checkpoint recovery U2OS cells were depleted for PHF3, BRD2, and ACTL6A and prepared for analysis by flow cytometry to determine checkpoint recovery (top panels) or lysed for Western blotting using the indicated antibodies (bottom panels). Error bars represent the SEM of three independent experiments. Statistical significance was determined using a two-tailed, unpaired t-test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).U2OS cells were locally irradiated using a 405nm laser and fixed directly thereafter for immunofluorescence. Alexa 488 represents PHF3, ACTL6A, or BRD2 staining. Scale bar is 5 µm.U2OS cells expressing YPF-BRD2 and mCherry-NBS1 were locally irradiated and followed by time-lapse imaging. Graph shows the quantifications of at least 30 cells analyzed in three individual experiments, and error bars represent the SD. Scale bar is 5 µm.
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