Figure 4

SK-N-AS cellular proliferation is insensitive to S6K1 or GLI1 overexpression. (A) SK-N-AS cells, cultured for 48 hours following transfection with control pCMV5 vector, and expression constructs for wild type S6K1 (S6K1 WT) constitutively activated S6K1 (S6K1T389E), function-loss S6K1 (S6K1T389A) and GLI1, were subjected to the EdU incorporation assay for 4 hours. (B) SK-N-AS cells, cultured for 48 hours following transfection with control siRNAs and pCMV5 vector (siCN + pCMV), S6K1 siRNAs and pCMV5 vector (siS6K1 + pCMV) and S6K1 siRNAs and GLI1 expression construct (siS6K1 + pGLI1), were subjected to the EdU incorporation assay for 4 hours. The data were analyzed with the one-way ANOVA test followed by Tukey’s multiple comparison using the GraphPad Prism software. Each bar represents the mean ± SEM of three independent experiments *, Statistical significant, P < 0.01 compared to control. One representative experiment is shown in the histographs. For both (A) and (B) the percentage of cells labeled with Alexa Fluor 488 azide was detected by flow cytometry. Note that overexpression of GLI1 can not rescue the reduced proliferation elicited by knocking down S6K1.