Figure 2.
PHF6 is recruited to the vicinity of DNA breaks for efficient DNA repair. (A) Representative confocal images showing PHF6 colocalization with γ-H2AX after UV micro-irradiation in U2OS cells. (B) Representative confocal images showing PHF6 colocalizing with γ-H2AX in a single double-strand break (DSB) induced by I-SceI expression in U2OS-DR GFP cells. (C) Upper panel, schematics of DSB induction after doxycycline and PHF6 recruitment to the vicinity of a DSB (region A) and to 2 different regions away from the DSB (regions B and C). Lower panel, quantification of ChIP assay showing PHF6 recruitment to the vicinity of the I-SceI DSB site in U2OS-DR-GFP cells in 3 different genomic regions (regions A-C) in 2 independent experiments. Bar graphs represent mean ± standard error of the mean. EV (empty-vector, not I-SceI-induced DSB control). (D) GFP percentage measured by flow cytometry in U2OS cells expressing 2 different short hairpin RNAs (shRNAs) targeting PHF6 or control shRNA containing integrated reporters to measure DNA repair efficiency through homologous recombination (U2OS-DR-GFP), single-strand annealing (SA-GFP), or non-homologous end-joining (EJ5-GFP). The percentage of GFP-positive cells is plotted as percent relative to the control cells. Data are representative of 4 independent experiments. Bar graphs represent mean ± standard error of the mean. (E) Representative images of Rad51 foci (red) obtained after 1- or 6-hour recovery from neocarzinostatin (NCS) treatment in control (EV, control sgRNA) and PHF6-knockout cells. DNA was stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bar, 25 μM. (F) Quantification of the intensity of Rad51 foci per cell in control (EV, control sgRNA) and PHF6-knockout U2OS cells. Between 100 and 200 cells were analyzed per condition. Statistical analysis was conducted by using a nonparametric Mann-Whitney test. Data are representative of 2 independent experiments. (G) Representative alkaline comet images performed in untreated U2OS cells or after NCS treatment (100 ng/mL) and recovery for 1, 4, or 6 hours in cells infected with a control shRNA (shControl) or a PHF6-targeting shRNA (shPHF6). (H) Dot plot showing individual percentages of comet tail DNA. The median value of >70 nuclei per experimental condition is indicated. Statistical analysis was conducted by using the Mann-Whitney test. Data are representative of 2 independent experiments. (I) Western blot showing the presence of phosphorylated γ-H2AX after recovery from irradiation (1 Gy) for the indicated times in PHF6 control or knock-out primary T-ALL cells. Gapdh is shown as loading control. Tmx, tamoxifen. (J) Analysis of apoptosis upon irradiation at 8 Gy in U2OS infected with control single guide RNA (sgRNA) or sgRNA#1/sgRNA#2. Bar graphs represent mean ± standard deviation (SD). (K) Quantification of ChIP assay showing CHD4 recruitment to the vicinity of the I-SceI DSB site in U2OS-DR-GFP cells (region A) in the presence (PHF6 shRNA '-') or absence of PHF6 (PHF6 shRNA '#7'). Data are representative of 3 independent experiments. EV (empty-vector, not I-SceI-induced DSB control). Bar graphs represent mean ± SD. (L) Quantification of ChIP assay showing SMARCB1 recruitment to the vicinity of the I-SceI DSB site in U2OS-DR-GFP cells (region A) in the presence (PHF6 shRNA '-') or absence of PHF6 (PHF6 shRNA '#7'). Data are representative of 3 independent experiments. EV (empty-vector, not I-SceI-induced DSB control). Bar graphs represent mean ± SD. (M) Left, western blot confirming endogenous PHF6 interaction by immunoprecipitation with SNF2H before and after treatment with 100 ng/mL NCS. Right: quantification of Phf6 levels normalized to Phf6 input ± NCS. All P values in the graphics were assessed by using a 2-tailed, unpaired Student’s t test. IgG, immunoglobulin G; n.s., not significant.

PHF6 is recruited to the vicinity of DNA breaks for efficient DNA repair. (A) Representative confocal images showing PHF6 colocalization with γ-H2AX after UV micro-irradiation in U2OS cells. (B) Representative confocal images showing PHF6 colocalizing with γ-H2AX in a single double-strand break (DSB) induced by I-SceI expression in U2OS-DR GFP cells. (C) Upper panel, schematics of DSB induction after doxycycline and PHF6 recruitment to the vicinity of a DSB (region A) and to 2 different regions away from the DSB (regions B and C). Lower panel, quantification of ChIP assay showing PHF6 recruitment to the vicinity of the I-SceI DSB site in U2OS-DR-GFP cells in 3 different genomic regions (regions A-C) in 2 independent experiments. Bar graphs represent mean ± standard error of the mean. EV (empty-vector, not I-SceI-induced DSB control). (D) GFP percentage measured by flow cytometry in U2OS cells expressing 2 different short hairpin RNAs (shRNAs) targeting PHF6 or control shRNA containing integrated reporters to measure DNA repair efficiency through homologous recombination (U2OS-DR-GFP), single-strand annealing (SA-GFP), or non-homologous end-joining (EJ5-GFP). The percentage of GFP-positive cells is plotted as percent relative to the control cells. Data are representative of 4 independent experiments. Bar graphs represent mean ± standard error of the mean. (E) Representative images of Rad51 foci (red) obtained after 1- or 6-hour recovery from neocarzinostatin (NCS) treatment in control (EV, control sgRNA) and PHF6-knockout cells. DNA was stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bar, 25 μM. (F) Quantification of the intensity of Rad51 foci per cell in control (EV, control sgRNA) and PHF6-knockout U2OS cells. Between 100 and 200 cells were analyzed per condition. Statistical analysis was conducted by using a nonparametric Mann-Whitney test. Data are representative of 2 independent experiments. (G) Representative alkaline comet images performed in untreated U2OS cells or after NCS treatment (100 ng/mL) and recovery for 1, 4, or 6 hours in cells infected with a control shRNA (shControl) or a PHF6-targeting shRNA (shPHF6). (H) Dot plot showing individual percentages of comet tail DNA. The median value of >70 nuclei per experimental condition is indicated. Statistical analysis was conducted by using the Mann-Whitney test. Data are representative of 2 independent experiments. (I) Western blot showing the presence of phosphorylated γ-H2AX after recovery from irradiation (1 Gy) for the indicated times in PHF6 control or knock-out primary T-ALL cells. Gapdh is shown as loading control. Tmx, tamoxifen. (J) Analysis of apoptosis upon irradiation at 8 Gy in U2OS infected with control single guide RNA (sgRNA) or sgRNA#1/sgRNA#2. Bar graphs represent mean ± standard deviation (SD). (K) Quantification of ChIP assay showing CHD4 recruitment to the vicinity of the I-SceI DSB site in U2OS-DR-GFP cells (region A) in the presence (PHF6 shRNA '-') or absence of PHF6 (PHF6 shRNA '#7'). Data are representative of 3 independent experiments. EV (empty-vector, not I-SceI-induced DSB control). Bar graphs represent mean ± SD. (L) Quantification of ChIP assay showing SMARCB1 recruitment to the vicinity of the I-SceI DSB site in U2OS-DR-GFP cells (region A) in the presence (PHF6 shRNA '-') or absence of PHF6 (PHF6 shRNA '#7'). Data are representative of 3 independent experiments. EV (empty-vector, not I-SceI-induced DSB control). Bar graphs represent mean ± SD. (M) Left, western blot confirming endogenous PHF6 interaction by immunoprecipitation with SNF2H before and after treatment with 100 ng/mL NCS. Right: quantification of Phf6 levels normalized to Phf6 input ± NCS. All P values in the graphics were assessed by using a 2-tailed, unpaired Student’s t test. IgG, immunoglobulin G; n.s., not significant.

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