Fig. 5

NEDD8 expression negatively regulates the activation of the Akt/mTOR signaling axis in OSCC cells. A–D Western blot analyses of the protein levels of phosphorylated Akt (p-Akt), Akt, p-mTOR, mTOR and GAPDH in the tested OSCC cell lines, HSC4 cell variants (B), HSC3 cell variants (C) and HSC4 cells treated with the indicated MLN4924 concentrations for 24 h (D). E and F Crystal violet staining (E) and histogram (F) for the results of three independent colony formation assays for nonsilencing control HSC4 cells and NEDD8-silenced HSC4 cells treated without (untreated, UT) or with 0.1% DMSO (Mock) and the indicated rapamycin (RAPA) concentrations for 24 h prior to treatment with irradiation at 4 Gy. The symbol “***” represents statistical significance at p < 0.001