Fig 2: OTSSP167 decreases GSCs proliferation and neurosphere formation by inhibiting the phosphorylation and transcriptional activation of FOXM1. (A, B) Effects of OTSSP167 on cell viability in GSC1 (A) and GSC2 (B). Cells treated with OTSSP167 were plated in triplicate wells in stem cell media, and then cell viabilities were assessed at the indicated times (day 0–day 12). In vitro limiting dilution assays of GSC1 (C) and GSC2 (D) treated with indicated does of OTSSP167 or 0.1% DMSO. (E–H) Effects of OTSSP167 on GSCs neurosphere formation. Representative images of GSC1 (E) and GSC2 (G) neurosphere and the quantification of sphere numbers of GSC1 (F) and GSC2 (H) treated with indicated doses of OTSSP167 or DMSO. (I) Immunoblot analysis of MELK, p-FOXM1(Ser35), FOXM1 and ß-actin in GSC1 treated with indicated doses of OTSSP167. (J) OTSSP167 inhibits the transactivation ability of FOXM1. Relative FOXM1 luciferase activity normalized with respect to corresponding renilla luciferase activity is shown. (K) The expression levels of FOXM1 were detected by western blot to check the FOXM1 cDNA plasmid transfection efficacy. (L) In vitro cell viability assay of FOXM1 overexpression in GSC1 cells treated with 25 nM OTSSP167 or the vehicle control. All the Data are presented as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the 0.1% DMSO treated group.