Figure 2. Resveratrol prevents rapamycin-induced induction of autophagy. (A) WT MEFs and TSC2^−/− MEFs were treated with 20 nM rapamycin and/or 100 µM resveratrol for 24 h. Cells were lysed, and protein levels of p62 and actin were analyzed by immunoblot. (B) TSC2^−/− MEFs were treated with 20nM rapamycin and/or 100 µM resveratrol, with or without 4-h pretreatment with 100 nM BafA1. Cells were lysed and blotted for LC3 and actin. The histogram shows LC3-II levels (lower band) normalized to actin levels. LC3-II and actin levels were quantified using Image Studio 3.1 (LI-COR), and plotted using Excel. (C) ELT3 TSC2 o/e and ELT3 cells were treated with 20 nM rapamycin and/or 100 µM resveratrol, with or without 4-h pretreatment with 100 nM BafA1. Cells were lysed and blotted for LC3 and actin. The histogram shows LC3-II levels (lower band) normalized to actin levels. LC3-II and actin levels were quantified using Image Studio 3.1 (LI-COR), and plotted using Excel. (D) 621–101 cells were treated with 20 nM rapamycin and/or 100µM resveratrol for 24 h. Cells were lysed and probed for p62 and α-tubulin levels. (E) 621–101 and 621–101 TSC2 o/e cells were treated with 20 nM rapamycin and/or 100 µM resveratrol for 24 h Cells were lysed and probed for p-S6 (T240/244) and actin levels. (F) 621–101 and 621–101 TSC2 o/e cells were treated as described in (B). Histograms show LC3-II levels (lower band) normalized to either actin or LC3-I levels (upper band).