Fig. 2

TGF-β2 promotes LC3 conversion with time- and dose-dependent effects. a Immunoblot analysis showed the dose-dependent effect of TGF-β2 treatment (24 h) on the endogenous conversion of LC3 in U251, T98 and U87 cell lines. The LC3B-II/I ratio (middle) was calculated using ImageJ 1.36b. b Time-dependent effects of (10 ng/ml) on U251, T98 and U87 cell lines. c U251, T98, and U87 cells stably that expressed GFP-LC3B from lentiviruses after TGF-β2 treatment was observed by fluorescence microscopy. The percentage of positive cells were quantified (scale bars 50 μm). d Transmission electron microscopy showed the formation of autophagosomes after TGF-β2 treatment (10 ng/ml, 24 h) in U251 cells. Bottom left, enlarged image. Representative images of autophagosomes are shown at the bottom (arrows). The number of autophagosomes per cell were quantified. e In primary cells, TGF-β2 could also initiate autophagy showed by western blot under the same condition of cell lines’ (10 ng/ml, 24 h). f TGF-β2-induced autophagy are not affected upon mTOR agonist 3-MA (25 ng/ml). And blocking the autolysosomal step of autophagy by Baf (10 ng/ml) showed up-regulation of SQSTEM/P62. Graph shows densitometric analysis of P62 and actin expressed as p62/actin ratio. g Microarray analysis showed autophagy-related gene expression levels in TGF-β2-treated U251 cell lines. (Student’s t-test; Columns, means of triplicate assays, **, P < 0.01; error bars are SD)