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. 2019 Feb 20;38(7):e101005. doi: 10.15252/embj.2018101005

Figure EV4. Cellular function of NBS1 in DNA end resection.

Figure EV4

  1. Western blot of Flp‐In T‐REx NBS1‐myc expressing cells. Expression of recombinant NBS1 was induced for 24 h with doxycycline (DOX) prior to extract preparation.
  2. Clonogenic survival assay showing that expression of siRNA‐resistant recombinant NBS1 rescues the radiosensitivity of NBS1‐depleted cells. Averages shown; n = 3, error bars, SD.
  3. Immunofluorescence experiment showing efficient depletion of NBS1 by siRNA and cytoplasmic localization of MRE11 in the NBS1‐depleted cells. Expression of myc‐tagged NBS1 restored MRE11 nuclear localization.
  4. Western blot showing efficient depletion of NBS1 and CtIP by siRNA transfection and expression of siRNA‐resistant myc‐tagged recombinant wt NBS1 and variants.
  5. NBS1 R28A and K160M mutations impair the interaction between NBS1 and CtIP in cell extracts. Flag‐CtIP and NBS1‐myc variants were overexpressed in 293‐T cells, where indicated. Anti‐FLAG affinity resin was used to pull down FLAG‐CtIP and interacting partners.
  6. Western blot showing the downregulation of CtIP and NBS1 upon siRNA treatment in the HR reporter assays in (G).
  7. HR repair reporter assay after CtIP and NBS1 depletion. Bars and error bars represent mean and SD of four independent experiments. Statistical significance was tested by unpaired t‐test (*P = 0.02).
  8. Western blot of extracts prepared from wild‐type U2OS cells, NBS1ΔN cells, and NBS1ΔN cells stably expressing mNeonGreen (mNG)‐tagged wild‐type (WT) and R28A K160M NBS1 mutant full‐length NBS1, either mock‐depleted (siCTRL) or depleted of CtIP (siCtIP) by siRNA. Note the presence of a presumably hypomorphic 40 kDa C‐terminal fragment of NBS1 (NBS1hm) in the NBS1ΔN cell line.
  9. Immunofluorescence of NBS1∆N U2OS cells stably expressing mNeonGreen (mNG)‐tagged wild‐type (WT) and R28A K160M mutant full‐length NBS1. Note that MRE11 nuclear localization is partially impaired in NBS1∆N cells, which is fully rescued by heterologous expression of NBS1 wt and R28A K160M mutant.
  10. Quantitation of resection activity as scored by RPA foci in wild‐type U2OS cells and NBS1∆N cells. At least 260 cells were assessed per condition. The horizontal line represents a median, the box spans from 25th to 75th percentile, and the whiskers from 10th to 90th percentile.