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. 2020 Oct 1;21(11):e48676. doi: 10.15252/embr.201948676

Figure 5. EWS regulates genomic integrity in a PARP1‐dependent manner.

Figure 5

  • A
    Relative cell viability of cells transfected with siCon (WT), siEWS (EWS KD), PARP1 (PARP‐1 KD), and double siRNA (DKD) after MMS treatment. Error bars represent as mean ± SEMs, and technical repeats (n = 3). Significance determined by two‐way ANOVA, ***P < 0.001. Analysis (***) indicates differences between DKD with EWS KD.
  • B
    Additional Parp1 knockout in Ews knockout cells (DKO) were subjected to viability test after MMS treatment. Error bars represent as mean ± SEMs, and technical repeats (n = 3). Significance determined by two‐way ANOVA, ***P < 0.001. *** indicated differences between DKO with EWS‐KO.
  • C
    After transfection of siCon, siEWS, PARP1, and double siRNA, the effect of co‐depletion of PARP1 and EWS on the γH2AX and phosphorylated CHK1 upon MMS treatment was analyzed by Western blot.
  • D
    After treatment of MMS, the effect of additional Parp1 knockout in Ews knockout cells (Double KO) to the γH2AX and phosphorylated CHK1 were analyzed using Western blot.
  • E
    Whole cell expression of PAR in mBA (WT: Ews‐WT, KO: Ews‐KO, DKO: double KO).
  • F
    Relative NAD+/NADH ratios were measured in WT, Ews −/− and DKO cells. Error bars represent as mean ± SEMs, and technical repeats (n = 3). Significance determined by two‐way ANOVA, ***P < 0.001.
  • G
    PARGi (5 μM, 24 h) treated WT and Ews −/− cells were subjected to cellular viability assay. Error bars represent as mean ± SEMs, and technical repeats (n = 3). Significance determined by two‐way ANOVA.
  • H
    Relative cellular viability was measured after NMN treatment, with or without MMS pretreatment, in WT and Ews −/− cells. Error bars represent as mean ± SEMs, and technical repeats (n = 3). Significance determined by two‐way ANOVA, ***P < 0.001.
  • I
    Chromatin‐bound PARP1 was quantified by Western blot in WT and Ews −/− cells treated with Olaparib (5 μM, 24 h) treatment.
  • J
    Relative viability was measured in WT and Ews −/− cells upon treatment of Olaparib for 24 h. Error bars represent as mean ± SEMs, and technical repeats (n = 3). Significance determined by two‐way ANOVA, ***P < 0.001.
  • K
    Table of Ews −/− pups survival number in control (normal water) or NMN supplements (NMN containing water) groups 3 days after birth. Statistics (χ2) were determined by chi‐square test.

Source data are available online for this figure.