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. 2018 Sep 4;128(10):4280–4296. doi: 10.1172/JCI120518

Figure 6. MDC1 is an essential mediator in the USP7-regulated DDR.

Figure 6

(A) HeLa cells stably expressing HA-ER-AsiSI were transfected with USP7 siRNA and cultured in the presence of 0.5 μM 4-OHT for 4 hours. Then, cells were fixed and immunostained with the indicated antibodies followed by confocal microscopic analysis. White arrows indicate cells with different amounts of USP7 depletion. Representative images from biological triplicate experiments are shown. Scale bars: 10 μm. (B) HeLa cells stably expressing control vector or FLAG-MDC1 were cotransfected with the indicated siRNAs and GFP-MRE11. Then, cells were subjected to UV laser micro-IR and live-cell imaging at the indicated time points. Representative images from biological triplicate experiments are shown. Scale bars: 10 μm. (C) Images shown are analogous to those in B, but for GFP-RAD50. Scale bars: 10 μm. (D) Images shown are analogous to those B, but for GFP-NBS1. Scale bars: 10 μm. (E) HeLa cells stably expressing HA-ER-AsiSI were transfected with the indicated siRNAs and control vector, FLAG-MDC1, or Myc-RNF168. Cells were then treated with 0.5 μM 4-OHT, fixed, and immunostained with antibodies against BRCA1 and USP7 followed by confocal microscopic analysis. Representative images from biological triplicate experiments are shown. Scale bars: 10 μm. (F) Images shown are analogous to those in E, but for 53BP1 and USP7. Scale bars: 10 μm. (G) Cellular extracts were collected from cells used in E and F, and expression of the indicated proteins was assessed by Western blotting.