Fig. 5

Autophagy markers induced in DMSC by MPA treatment. a Endogenous LC3-I and LC3-II levels in DMSC in untreated and after MPA treatment. DMSC were cultured for the indicated times, and total lysates subjected to immunoblot analysis using anti-LC3 antibody and anti-tubulin antibody. LC3-I and LC3-II positions are indicated. b To prevent lysosomal degradation of LC3-II, 30 µg/ml of chloroquine (CQ) was added to the medium where indicated. The increase in LC3-II as a consequence of the addition of the lysosomal inhibitor would indicate that the observed decrease in LC3-II in MPA treated DMSC would be due to an increased autophagic flux. c Lysates from MPA-treated cells for 72 h were subjected to immunoblot analysis for three indicators of autophagy: p62, whose accumulation is among the best characteristic of autophagy-deficient tissues, and the phosphorylated forms of mTOR and p70-S6K, which negatively regulate autophagy