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. 2019 Nov 29;21(1):e48460. doi: 10.15252/embr.201948460

Figure 3. PHF6 regulates G2 checkpoint recovery.

Figure 3

  • A
    Graphical explanation of the effect of PHF6 on checkpoint recovery through DNA repair and p53.
  • B, C
    U2OS 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 βTrCP 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, E
    U2OS 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, G
    U2OS 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 γH2AX 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).
  • H
    U2OS 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).