Figure S3.
c-NHEJ is required for DMSR. (A) The efficiency of ATR inhibitor or ATM inhibitor was confirmed by probing with pS345-CHK1 antibody or pS1981-ATM antibody, respectively. (B) The time course of DNA-PK activation after RPE-1 cells were exposed to 5 Gy IR in G1/S. DNA-PK activation was determined by immunoblotting with anti-pS2056 DNA-PKcs antibody. (C) Left: Quantitative RT-PCR shows the knockdown efficiency of indicated genes. Right: Western blot analysis shows the efficiency of indicated siRNAs. RPE-1 cells were transfected with indicated siRNAs. Cells were then collected and extracted after 48 h of transfections. The efficiency was measured by indicated antibodies. (D) Depletion of CtIP or other proteins responsible for end resection, such as EXD2, EXO1, and NBS1, at G1 phase does not affect DMSR. U2OS cells were transfected with indicated siRNAs and synchronized at G1/S phase. Cells were then exposed to 2 Gy IR and recovered for 4 h. Left: Western blot analysis shows the efficiency of indicated siRNAs. Right: Microtubule nucleation ability was determined by microtubule regrowth assay. Quantitation of microtubule length was assayed as in Fig. 1. This experiment and the experiment in Fig. 4 B belong to a same group of experiments. Thus, the same data for the control group was included in this figure and Fig. 4 B. (E) DMSR is significantly inhibited by depletion of 53BP1 or Ku70. U2OS cells were transfected with indicated siRNA and synchronized at G1/S phase. Cells then were exposed to 2 Gy IR and recovered for 4 h. Microtubules were stained with anti–β-tubulin antibody. Scale bar, 20 µm. (F) Western blot analysis shows the efficiency of indicated siRNAs. (G) Depletion of Ligase 4, XRCC4, or XLF, which are required for c-NHEJ, promote DMSR in U2OS cells. Microtubules (green) were stained with anti–β-tubulin antibody. Scale bar, 20 µm. (H) Western blot analysis shows the efficiency of indicated siRNAs. (I) The activation of DDR and kinetics of DNA damage repair in IR-treated U2OS control cells or siLig4 cells was determined by γH2AX foci formation (n > 100). ****, P < 0.0001; MT, microtubule; UT, untreated.

c-NHEJ is required for DMSR. (A) The efficiency of ATR inhibitor or ATM inhibitor was confirmed by probing with pS345-CHK1 antibody or pS1981-ATM antibody, respectively. (B) The time course of DNA-PK activation after RPE-1 cells were exposed to 5 Gy IR in G1/S. DNA-PK activation was determined by immunoblotting with anti-pS2056 DNA-PKcs antibody. (C) Left: Quantitative RT-PCR shows the knockdown efficiency of indicated genes. Right: Western blot analysis shows the efficiency of indicated siRNAs. RPE-1 cells were transfected with indicated siRNAs. Cells were then collected and extracted after 48 h of transfections. The efficiency was measured by indicated antibodies. (D) Depletion of CtIP or other proteins responsible for end resection, such as EXD2, EXO1, and NBS1, at G1 phase does not affect DMSR. U2OS cells were transfected with indicated siRNAs and synchronized at G1/S phase. Cells were then exposed to 2 Gy IR and recovered for 4 h. Left: Western blot analysis shows the efficiency of indicated siRNAs. Right: Microtubule nucleation ability was determined by microtubule regrowth assay. Quantitation of microtubule length was assayed as in Fig. 1. This experiment and the experiment in Fig. 4 B belong to a same group of experiments. Thus, the same data for the control group was included in this figure and Fig. 4 B. (E) DMSR is significantly inhibited by depletion of 53BP1 or Ku70. U2OS cells were transfected with indicated siRNA and synchronized at G1/S phase. Cells then were exposed to 2 Gy IR and recovered for 4 h. Microtubules were stained with anti–β-tubulin antibody. Scale bar, 20 µm. (F) Western blot analysis shows the efficiency of indicated siRNAs. (G) Depletion of Ligase 4, XRCC4, or XLF, which are required for c-NHEJ, promote DMSR in U2OS cells. Microtubules (green) were stained with anti–β-tubulin antibody. Scale bar, 20 µm. (H) Western blot analysis shows the efficiency of indicated siRNAs. (I) The activation of DDR and kinetics of DNA damage repair in IR-treated U2OS control cells or siLig4 cells was determined by γH2AX foci formation (n > 100). ****, P < 0.0001; MT, microtubule; UT, untreated.

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