Inhibitory effect of SPP-ARV-825 on GL261 cells proliferation in vitro. (A) Cell viabilities of GL261 cells treated with ARV-825, PP-ARV-825 and SPP-ARV-825 at different concentrations for 48 h (n = 3). (B) Representative images of colony formation of GL261 cells incubated with 200 ng/mL ARV-825, PP-ARV-825 and SPP-ARV-825. (C) Quantification of colony formation (n = 3). (D) GL261 cells cycle distribution determined by flow cytometry after treatment with 400 ng/mL ARV-825, PP-ARV-825 and SPP-ARV-825 for 24 h. (E) Quantification of cell cycle distribution (n = 3). (F) Flow cytometry detection of GL261 cells proliferation after treatment with 400 ng/mL SPP-ARV-825 for 24 and 48 h. (G) Quantification of Edu positive GL261 cells (n = 3). (H) BRD4 expression detection by Western blot analysis after incubation of GL261 cells with different concentrations of SPP-ARV-825 for 48 h and with 400 ng/mL SPP-ARV-825 for different time. (I, J) Representative Western blot analysis of (I) cyclin D1, cyclin E1 and β-action and (J) P-AKT, AKT, P-ERK1/2, ERK1/2, P-STAT3, STAT3 and GAPDH in GL261 cells treated with different concentrations of SPP-ARV-825 for 24 h. GAPDH or β-action served as a loading control. Data are presented as mean ± SEM. ∗P < 0.05, ∗∗∗P < 0.001 and ∗∗∗∗P < 0.0001.