Fig. 5: Pontin functions as a transcription co-activator of E2F1 in GBM cells.
From: The ATPase Pontin is a key cell cycle regulator by amplifying E2F1 transcription response in glioma
A Co-IP using IgG or E2F1 antibody in U87MG and U251 cell extracts. Endogenous Pontin and Reptin were pulled-down and detected in the pulled-down fractions by immunoblot. B Co-localization of endogenous Pontin (green) and E2F1 (red) in U251 cells. Representative images from biological triplicate experiments are shown. Scale bar, 10 μm. C Top: Schematic diagram of Pontin domains and construction of Pontin domain deleting mutants. All mutants are flag tagged. Bottom: Co-IP using the flag antibody in U87MG cell extracts. Interactions between flag tagged Pontin proteins and endogenous E2F1 were confirmed by immunoblot. D Luciferase assay was performed with reporters driven by AURKA promoter containing two E2F1-binding elements (AURKA −1700) or deleting E2F1-binding region (AURKA –255). Effect of Pontin or the Pontin ΔATPase (the ATPase domain deficient mutant) expression on AURKA –1700 or AURKA –255 promoter-luciferase activity in the presence or absence of E2F1 was shown. Luciferase activities were normalized by β-galactosidase activity. Values are expressed as mean ± SD of three independent experiments. *P < 0.05; ***P < 0.001. E Western blot analyses of Pontin, CDK1, and CDK4 expressions in stable U87MG sub-cell lines (sh-NC, sh-Pontin) transfected with indicated plasmids. F The growth curves of the indicated cells assessed by MTS assays. Data are presented as the mean ± SD. ***P < 0.001.
