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. 2017 Mar 23;18(5):745–764. doi: 10.15252/embr.201643191

Figure 7. NELF‐E promotes HDR and NHEJ of DSBs.

Figure 7

  1. Western blot shows NELF‐E knockdown using two different shRNAs (#2 and #4). β‐actin was used as a loading control.
  2. Colony formation assay shows that NELF‐E‐deficient cells are hypersensitive to IR. The numbers of colonies were normalized to the percentage of undamaged cells and plotted as a function of IR dosage. Error bars represent SEM (n = 4). Two‐way ANOVA was used to test for differences at each dose.
  3. Western blot shows depletion of NELF‐E and Rad51 in U2OS‐HR‐ind cells transfected with NELF‐E and Rad51 siRNAs. β‐actin and histone H3 were used as loading controls.
  4. NELF‐E promotes HDR of DSBs induced by I‐SceI endonuclease. To measure the integrity of HDR, U2OS‐HR‐ind cells were transfected with two different NELF‐E siRNAs and the efficiency of HDR was determined using flow cytometric analysis. For controls, U2OS‐HR‐ind cells were transfected with either control siRNA or Rad51 siRNA. Results shown are typical of three independent experiments. Error bars represent SD. P‐values were calculated by two‐sided Student's t‐test relative to Ctrl siRNA, **P < 0.01, ***P < 0.001.
  5. Western blot shows depletion of NELF‐E in HeLa‐pEJSSA cells transfected with NELF‐E and Ku80 siRNAs. β‐actin and histone H3 were used as loading controls.
  6. NELF‐E depletion affects the integrity of NHEJ of DSBs. NELF‐E‐depleted HeLa cells containing pEJSSA plasmid stably integrated into their genome were used to determine the efficiency of NHEJ as described in the Materials and Methods. For controls, HeLa‐pEJSSA cells were transfected with either control or Ku80 siRNA. Results shown are typical of three independent experiments. Error bars represent SD. P‐values were calculated by two‐sided Student's t‐test relative to Ctrl siRNA, **P < 0.01, ***P < 0.001.