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

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© 2018 Stolte et al.

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Figure 9. — Loss of PPM1D and USP7 is rescued by concurrent TP53 loss. (A) Western blots showing attenuated increase of p53 protein levels in TC32, TC138, and CHLA258 cells infected with sgRNAs targeting TP53 after etoposide treatment (Control, control sgRNA; sg #1, sgTP53 #1; sg #2, sgTP53 #2; sg #4, sgTP53 #4; sg #5, sgTP53 #5). Cells were treated with vehicle or 50 μM etoposide for one hour (Veh, vehicle; Eto, etoposide). (B) TP53 knockout cells were treated with ATSP-7041 for 3 d. Values were normalized to vehicle controls. Each data point shows the mean of eight replicates; error bars are mean values ± standard deviation. The experiment was performed twice and data points of one representative experiment are shown. (C) TP53 knockout cells were treated with GSK2830371 for 3 d. Values were normalized to vehicle controls. Each data point shows the mean of eight replicates; error bars are mean values ± standard deviation. The experiment was performed twice, and data points of one representative experiment are shown. (D) TP53 knockout cells were treated with P5091 for 3 d. Values were normalized to vehicle controls. Each data point shows the mean of eight replicates; error bars are mean values ± standard deviation. The experiment was performed twice, and data points of one representative experiment are shown. (E) Western blots showing decreased protein levels of USP7 after infection with sgRNAs targeting USP7 in TC32 TP53 knockout cells. (F) Western blots showing decreased protein levels of Wip1 after infection with sgRNAs targeting PPM1D in TC32 TP53 knockout cells. (G) Relative viability of TC32 TP53 knockout cells infected with sgRNAs targeting USP7 or PPM1D 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: not significant (n.s.) for P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. (H) Ewing sarcoma cells were treated with XL-188 for 3 d. TP53 wild-type Ewing sarcoma cell lines are shown in red. TP53 mutated Ewing sarcoma cell lines are shown in black. Values were normalized to vehicle controls. Each data point shows the mean of eight replicates; error bars are mean values ± standard deviation. The experiment was performed twice, and data points of one representative experiment are shown. (I) TP53 knockout cells were treated with XL-188 for 3 d. Values were normalized to vehicle controls. Each data point shows the mean of eight replicates; error bars are mean values ± standard deviation. The experiment was performed twice and data points of one representative experiment are shown.