FIGURE 2 from Antitumor Effects of PRMT5 Inhibition in Sarcomas

Antitumor effects of PRMT5 inhibition on STSs. A, Assessment of cell viability with PRMT5 inhibitor GSK3226595 (GSK595) in seven STS cell lines (with TP53 deleted or not) as determined by MTT assay (n = 3 or more). Cells were treated with a range of increasing concentrations of drug for 10 days and the IC₅₀ was calculated with GraphPad Prism software. B, Western blot analysis showing of SDMA, p53, and p21 protein expression in four STS cell lines after 10 days of GSK595 treatment at the respective IC₅₀. C, Quantitation of p53 and p21 bands in B using ImageJ software. GAPDH served as the loading control (n = 3). D, Proliferation assay after PRMT5 inhibition: Cells were untreated or treated with GSK595 at the IC₅₀ and counted after 3, 6, and 10 days by flow cytometry. Fresh medium with drug treatment at the appropriate concentration was used to replace the medium every 3 days (n = 3; **, P < 0.01; ***, P < 0.001 two-way ANOVA and Bonferroni multiple comparisons test). E, Clonogenic assay: Representative images of stained cells (crystal violet) after 10 days of GSK595 treatment at the IC₈₀ (n = 2). F, Efficacy of the PRMT5 inhibitor in vivo in IB115 xenograft. IB115 cells were used to establish xenograft tumors in NSG mice (n = 9 mice per group), and the tumors were treated with vehicle or GSK595 at 100 mg/kg orally twice daily (5 days-on/2 days-off; ***, P < 0.001 two-way ANOVA and Bonferroni multiple comparisons tests, arrow = start of treatment). Tumor growth, body weight, and final tumor weight were measured. G, Efficacy of the PRMT5 inhibitor in vivo in IB111 xenograft mice. IB111 cells were used to establish xenograft tumors in NSG mice, which were treated with the same concentrations as the IB115 cells. Tumor growth and body weight were measured, and the tumors were monitored until the end of the treatment period or until they reached 2,000 mm³ (day of sacrifice); the data were used to draw the survival curve.