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. 2019 Oct 15;38(21):e102361. doi: 10.15252/embj.2019102361

Figure EV2. The p97–ATX3 complex regulates RNF8 turnover at sites of DNA damage.

Figure EV2

  1. Western blot analysis of CHX chase kinetics showing accelerated RNF8 degradation in soluble fraction (cytoplasm + nucleoplasm) of siATX3‐depleted HeLa cells.
  2. Graph represents the quantifications of (A) (****P < 0.0001; two‐way ANOVA, n = 3, mean + SEM).
  3. ATX3 and RNF8 mRNA expression level analysed by quantitative PCR after indicated siRNA treatment in U2OS cells. The experiment was performed in quadruplet (n = 1) and columns represent the mean + SEM.
  4. Representative IF micrographs of U2OS cells showing recruitment of p97 or ATX3 to UV‐A micro‐laser‐induced DNA damage tracks. Scale bar: 10 μm.
  5. Representative IF images showing the UV‐A micro‐laser‐induced DNA damage tracks in U2OS cells. Endogenous RNF8 and γ‐H2AX signal at damage tracks after 30 min and 5 h of damage induction under indicated siRNA‐depleted conditions. Scale bar: 10 μm.
  6. Quantification of (E) at 30 min time point. Graph represents the average intensity of the RNF8 signal (***P < 0.001; unpaired t‐test, n = 3, mean + SEM, in average, at least 70 nuclei per condition and experiment).
  7. Quantification of (E) at 5‐h time point. Graph represents the average intensity of the RNF8 signal (*P < 0.05, ***P < 0.001; unpaired t‐test, n = 3, mean + SEM, in average, at least 70 nuclei per condition and experiment).
  8. Quantification of endogenous RNF8 signal intensity in HeLa cells at UV‐A micro‐laser‐induced DNA damage tracks 30 min and 5 h after damage induction under indicated siRNA‐depleted conditions. A second, commercially available (Proteintech) RNF8 antibody was used. Graph represents the average intensity of RNF8 signal (ns P > 0.05, **P < 0.01, ***P < 0.001; unpaired t‐test, n = 1, mean + SEM, more than 50 nuclei were analysed per condition and experiment).
  9. Representative IF micrographs of HeLa cells showing Flag‐RNF8 signal intensity at UV‐A micro‐laser‐induced DNA damage tracks 30 min after damage induction under indicated siRNA‐depleted conditions. Scale bar: 10 μm.
  10. Quantification of (I). Graph represents the average intensity of RNF8 signal (***P < 0.001; unpaired t‐test, n = 1, mean + SEM, more than 50 nuclei were analysed per condition and experiment).
  11. Graph represents the recruitment kinetics of GFP‐RNF8 at sites of two‐photon laser‐induced DNA damage spot in living U2OS cells under indicated conditions (ns P > 0.05, **P < 0.01; unpaired t‐test on area under curve, n = 2, for siNpl4 n = 1, mean + SEM, in average, at least five nuclei per condition and experiment).
  12. Graph represents the recruitment kinetics of GFP‐RNF8 at sites of two‐photon laser‐induced DNA damage spot in living U2OS cells under indicated conditions (**P < 0.01; unpaired t‐test on area under curve, n = 3, mean + SEM, in average, at least five nuclei per condition and experiment).
  13. Western blot analysis showing depletion efficiency of indicated siRNAs in U2OS cells.
  14. Graph represents the quantification of GFP (RNF8) foci in fixed U2OS cells after 30 min of IR (2 Gy) treatment, under indicated conditions (*P < 0.05, ****P < 0.0001; unpaired t‐test, n = 2, +SEM).

Source data are available online for this figure.