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. 2019 Apr 8;129(5):2043–2055. doi: 10.1172/JCI126414

Figure 5. Ablation of USP9X expression impairs the self-renewal, tumorigenicity, and radio/chemoresistance of MES GSCs.

Figure 5

(A and B) Primary neurosphere formation was assessed in MES 21 and 505 GSCs transduced with shCtrl or shUSP9X, reconstituted with vector control or ALDH1A3. Representative images are shown (A). Neurosphere formation efficiency (spheres/cells plated) was quantified. (B) Data are represented as means ± SD of 3 independent experiments. ***P < 0.001, 2-tailed Student’s t test. (C) In vitro limiting dilution sphere-forming frequency of MES 21 and 505 GSCs transduced with shCtrl or shUSP9X, reconstituted with vector control or ALDH1A3. Stem cell frequencies were estimated as the ratio 1/x with the upper and lower 95% confidence intervals, where 1 = stem cell and x = all cells. (D) IB analysis of USP9X, ALDH1A3, CD44, C/EBPβ, TAZ, p-STAT3, STAT3, VEGF-A, and c-MET levels in MES 21 and 505 GSCs expressing shCtrl or shUSP9X, reconstituted with vector control or ALDH1A3. (E) H&E-stained brain sections from mice intracranially implanted with MES 21 or 505 GSCs with indicated modifications. Red arrows indicate tumors. (F) Kaplan-Meier survival curves of mice intracranially injected with MES 21 or 505 GSCs with indicated modifications (n = 8). ****P < 0.0001, log-rank (Mantel-Cox) test. Scale bars: 500 μm (A); 1 mm (E).