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. 2018 Aug 6;215(8):2137–2155. doi: 10.1084/jem.20171066

Figure 2.

Figure 2.

Validation of MDM2 and MDM4 as dependencies in TP53 wild-type Ewing sarcoma. (A) Western blots demonstrate abrogation of the observed increase in MDM2 protein levels upon RG7388 treatment (1 µM; 4 h) in TP53 wild-type cell lines TC32 and TC138 cells infected with sgRNAs targeting MDM2 compared with a nontargeting control sgRNA and no response to RG7388 treatment in TP53 mutated cell lines A673 and EWS502. (B) Western blot demonstrating decreased protein levels of MDM2 with sgRNAs targeting MDM2 compared with a control guide in the SJSA-X cell line. (C) Relative viability of Ewing sarcoma and SJSA-X cells infected with sgRNAs targeting MDM2 compared with control sgRNAs 14 d after infection. Each data point shows the mean of eight replicates, and data are plotted as mean values ± standard deviation. The experiment was performed twice and data points of one representative experiment are shown. (D) Western blots showing decreased protein levels of MDM4 after infection with sgRNAs targeting MDM4 compared with control sgRNAs. (E) Western blot demonstrating decreased protein levels of MDM4 with sgRNAs targeting MDM4 compared with control guides in the SJSA-X cell line. (F) Relative viability of Ewing sarcoma and SJSA-X cells infected with sgRNAs targeting MDM4 or control sgRNAs 14 d after infection. Each data point shows the mean of eight replicates, and data are plotted as mean values ± standard deviation. The experiment was performed twice and data points of one representative experiment are shown. Significance was calculated by paired, two-tailed t test: n.s., not significant for P > 0.05; *, P ≤ 0.05; **, for P ≤ 0.01; ***, P ≤ 0.001).