Fig. 4.

a SRT2183 induces autophagy in glioma cells. LN229 and U87MG cells were treated with vehicle or 10 μM SRT2183 for 2, 4, 8, 12, 24 h, expression levels of LC3 and GAPDH were measured by IB analysis. b LN229 and U87MG cells were pre-transfected with GFP-LC3,after 24 h the cells were treated with vehicle or 10 uM SRT2183 for 24 h. The results were observed by confocal microscope. c LN229 and U87MG cells were treated with vehicle or SRT2183 for 12, 24 and 48 h. Expression levels of LC3, P-Akt (473), P-Akt (308), Akt, P-mTOR, and mTOR were analyzed by IB. GAPDH was used as a control for equal loading. d LN229 and U87MG cells were transfected with HA-Vector, HA-Akt, and myr-Akt for 24 h, then they were treated with vehicle or SRT2183 for 24 h. Expression levels of P-Akt (473) and LC3 were analyzed by IB. e LN229 and U87MG cells were treated with vehicle or SRT2183 following pre-treatment with either autophagy inducers BEZ235 and Rapamycin (Rapa), or autophagy inhibitors Bafilomycin A1 (BafA1), Chloroquine (CQ), and SAR405, or PI3K inhibitors LY294002 and Wortmannin (Wort). CCK8 analysis was employed to determine cell growth inhibition. The above experiments were performed three times with comparable results. Results portrayed as the mean ± SEM (***p < 0.001).