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

Figure EV3. ATX3 operates downstream of RNF8 and K63‐Ub at sites of DNA damage.

Figure EV3

  1. Western blot analysis of GFP‐ATX3 Co‐IP in HEK293 cells showing interaction of ATX3 with endogenous RNF8 but not with MDC1 under physiological conditions and after IR (10 Gy).
  2. Representative IF images showing kinetics of endogenous MDC1 foci after 2 Gy of IR treatment in U2OS‐WT and U2OS‐∆ATX3 cells (scale bar: 10 μm).
  3. Quantification of (B). Graph representing percentage of nuclei with > 5 MDC1 foci, measured in more than 100 cells per condition per experiment (ns P > 0.05, **P < 0.01; unpaired t‐test, n = 2, mean + SEM).
  4. Western blot analysis of EGFP‐MDC1 denaturing‐IP showing phospho‐MDC1 (pSQ/TQ‐MDC1; RNF8 recruitment motif) signal in HEK293 and HEK293∆ATX3 cells after 10 Gy of IR treatment.
  5. Representative IF images showing the UV‐A micro‐laser‐induced DNA damage tracks in U2OS cells. Endogenous K63‐Ub and γ‐H2AX signal at damage tracks under indicated siRNA‐depleted conditions. Scale bar: 10 μm.
  6. Quantification of (E). Graph represents the average intensity of K63‐Ub (***P < 0.001; unpaired t‐test, n = 3, mean + SEM, at least 100 nuclei per condition and experiment).
  7. Representative IF images showing Flag‐RNF8‐WT or Flag‐RNF8‐RING* variant signal at UV‐A micro‐laser‐induced DNA damage tracks in U2OS cells after 30 min and 5 h of damage induction. Scale bar: 10 μm.
  8. Quantification of (G). Graph represents the average intensity of the RNF8 signal (*P < 0.01, ***P < 0.001; unpaired t‐test, n = 2, mean + SEM, at least 50 nuclei per condition and experiment).

Source data are available online for this figure.