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. 2020 Feb 21;39(7):e102668. doi: 10.15252/embj.2019102668

Figure EV3. SMCHD1 stimulates c‐NHEJ and promotes DNA damage signaling at TRF2‐depleted telomeres.

Figure EV3

  1. Western Blot detection of ATM pS1981, SMCHD1, MRE11, TRF2, and γH2AX, in HeLa cells transfected with the indicated shRNA plasmids (shTRF2, shMRE11, sh1 SMCHD1, sh2 SMCHD1, shTRF2/shMRE11, shTRF2/sh1 SMCHD1, shTRF2/sh2 SMCHD1) and empty vector (EV) control.
  2. Metaphase spreads from HeLa cells transfected with the indicated shRNA plasmids (shTRF2, shMRE11, sh1 SMCHD1, sh2 SMCHD1, shTRF2/shMRE11, shTRF2/sh1 SMCHD1, and shTRF2/sh2 SMCHD1) and empty vector (EV) control. Telomeric signals were detected with Cy3‐(CCCTAA)3 and are false‐colored in red. Scale bar: 5 µm.
  3. Quantification of telomere fusions in HeLa cells transfected with the indicated shRNAs and EV control. Bars represent average numbers of chromosome ends fused in three independent experiments with SDs. **P < 0.01; *P < 0.05 unpaired two‐tailed Student's t‐test.
  4. Representative images for detection of 53BP1 at telomeres in HeLa cells transfected with the indicated shRNA plasmids. Immunofluorescence (IF) for 53BP1 (yellow) was combined with telomeric (CCCTAA)3‐FISH (red), and the DNA was stained with DAPI. Scale bars: 5 µm.
  5. Quantification of the number of cells containing > 5 telomere dysfunction‐induced foci (TIFs) detected as in (A). Data represent mean of three independent experiments ± SD (> 100 cells/condition/experiment). *P < 0.05, **P < 0.01, unpaired two‐tailed Student's t‐test.

Source data are available online for this figure.