Fig 1: PHF2 knockdown alters 53BP1 and BRCA1 focus dynamics in response to IR. (A) U2OS cells were depleted for Luciferase (Luc), CtIP or PHF2 by siRNA. After 48 h, cells were treated with IR (10 Gy) and fixed after 1, 4 or 7 h. 53BP1 focus formation was analysed by immunofluorescence. Left panel: representative images. Right panel: quantification of three independent experiments with each at least 100 cells. (B) U2OS Rap80 knockout cells were depleted for Luc or PHF2. Cells were treated with IR (10 Gy), fixed at the indicated time points and ?H2AX (positive control for DNA damage induction) and BRCA1 focus formation was analysed as in (A). Left panel: representative images (IR, 4 h). (C) U2OS cells depleted for Luc or PHF2 were subjected to different DNA damaging agents and lysed after 1 h. Extracts were analysed by western blot using the indicated antibodies. (D) U2OS cells were transfected as in (C), treated with CPT or ETP and analysed by western blot with the indicated antibodies. (E) U2OS cells were depleted for CtIP or PHF2 by siRNA. Cells were treated with IR (3 Gy), fixed at the indicated time points and RPA2 focus formation was analysed as in (A). Left panel: representative images (IR, 1 h).
Fig 2: Pyridostatin promotes the formation of 53BP1-positive puncta in primary neurons(A) Primary cortical neurons were transfected with GFP and mApple-53BP1trunc constructs, and then treated with a vehicle (left panel; control) or with 1 µM pyridostatin (right panel; PDS). Neurons were imaged with an automated microscope every 24 h for 3 days. Scale bar is 5 µm. (B) Quantification of the mApple-53BP1trunc puncta index from (A) at different times. The puncta index was estimated by measuring the standard deviation of the 53BP1 fluorescence intensity. Note that 53BP1 puncta index is higher in pyridostatin-treated neurons than control neurons. *p<0.01, **p<0.001, and ***p<0.0001 (t-test). A.u., arbitrary units. Two hundred neurons were analyzed from two independent experiments. (C) Primary cortical neurons were treated with a vehicle (upper panel; control) or with 1 µM pyridostatin (bottom panel; PDS) overnight, fixed, and stained for MAP2c (red), a marker of DNA damage 53BP1 (green), and with the nuclear Hoechst dye (blue). Scale bar is 10 µm. (D) Quantification of the 53BP1 puncta index from (C). Pyridostatin (PDS) increased the 53BP1 puncta index compared to control neurons (cont). ***p<0.0001 (t-test). A.u., arbitrary units. Three hundred neurons were analyzed from three independent experiments.
Fig 3: PHF6 regulates G2 checkpoint recovery AGraphical explanation of the effect of PHF6 on checkpoint recovery through DNA repair and p53.B, CU2OS cells were transfected with four different siRNA oligonucleotides targeting PHF6, synchronized in G2, and subsequent recovery after 5 Gy of IR and the addition of nocodazole were analyzed by flow cytometry with MPM2 staining (B), or cells were lysed and analyzed using Western blotting with the indicated antibodies (C). PPM1D and ßTrCP were used as positive controls. 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).D, EU2OS WT, PHF6 knockout, and PHF6 knockout cells that were reconstituted with GFP-PHF6 were synchronized in G2, and recovery was determined after 2 Gy of IR using flow cytometry with pHH3 staining (D), or cells were lysed and analyzed using Western blotting with the indicated antibodies (E). Error bars represent the SEM of three independent experiments. Statistical significance was determined using a two-tailed, unpaired t-test (**P < 0.01). The remaining PHF6 signal in the PHF6 KO sample (E) is likely due to a crossreaction at similar height as PHF6 (also see Fig EV3C).F, GU2OS WT and PHF6 knockout cells were irradiated using 3 Gy of IR, and cells were fixed at different time points for immunofluorescence. In (F) is presented 53BP1 IRIF intensity. In (G) is shown the number of ?H2AX foci per cell. Error bars represent the SD of three independent experiments. Statistical significance was determined using a two-tailed, unpaired t-test (****P < 0.0001).HU2OS WT and PHF6 knockout cells were left untreated or irradiated with 3 Gy and processed after 1 h for comet assay analysis. The comet tail moment was analyzed in at least 50 individual cells. Error bars represent the SD of three independent experiments. Statistical significance was determined using a two-tailed, unpaired t-test (*P < 0.05, ****P < 0.0001).
Fig 4: PHF6 knockout cells display a similar phenotype as cells depleted for PHF6 A, BRPE1 cells were depleted of PHF6 using four different siRNA oligonucleotides. Checkpoint recovery after 5 Gy was analyzed by staining for MPM2 and flow cytometry (A), or cells were lysed and analyzed using Western blotting with the indicated antibodies (B). Error bars represent the SEM of three independent experiments. Statistical significance was determined using a two-tailed, unpaired t-test (***P < 0.001, ****P < 0.0001).CU2OS WT and three different PHF6 knockout clones were lysed and analyzed by Western blotting with the indicated antibodies.DU2OS WT and PHF6 knockout cells were irradiated at 2 Gy and fixed for immunofluorescence at 0, 1, and 16 h. Samples were stained with the indicated antibodies. Scale bar is 10 µm.EQuantification of number of 53BP1 IRIF per cell of (D) in which the error bars represent the SD of three independent experiments. Statistical significance was determined using a two-tailed, unpaired t-test (**P < 0.01, ****P < 0.0001).FU2OS cells were depleted of PHF6 with three different siRNA oligonucleotides. Cells were fixed, stained with propidium iodide, and analyzed by flow cytometry. Cell cycle distribution was determined using Flowlogic software. Error bars represent the SD of three independent experiments.GU2OS WT and two different PHF6 knockout cell lines were fixed, stained with propidium iodide, and analyzed by flow cytometry. Cell cycle distribution was determined using Flowlogic software. Error bars represent the SD of three independent experiments.
Fig 5: miR-7 regulates TP53BP1 through SP1. The (A and B) mRNA and (C and D) protein levels of TP53BP1 in A549 and SK-MES-1 cells are higher with upregulation of SP1 or downregulation of miR-7, and are lower with downregulation of SP1 or upregulation of miR-7 compared with the negative control. (E) The correlation coefficient (Spearman's test) between SP1 and TP53BP1 in LUAD, LUSC, and lung tissue from GEPIA, revealed that SP1 was correlated with the TP53BP1 in lung cancer tissue. *P<0.05, **P<0.01. miR-7, microRNA-7; TP53BP1, tumor suppressor p53-binding protein 1; SP1, specific protein 1; LUADC, lung adenocarcinoma; LUSC, lung squamous cell carcinoma.
Supplier Page from Abcam for Anti-53BP1 antibody