Fig. 2

HG increased autophagy in ADR-treated cells. (a) RKO stably transfected with GFP-LC3 plasmid were kept in low glucose (LG) or high glucose (HG) medium for 24 h and then treated with ADR (2 μg/ml) for 16, in the presence or absence of autophagy inhibitor chloroquine (CQ, 25 μM) for 4 h before observation to count GFP-LC3 puncta under fluorescence microscopy. Green indicates GFP-LC3. One of 10 representative micrographs is shown. (b) The relative number of GFP-LC3-positive cells was calculated from 10 random fields. The data are presented as the means ± S.D. from three independent experiments. (c) RKO cells were kept in low glucose (LG) or high glucose (HG) medium for 24 h and then treated with ADR (2 μg/ml) for 16 h in the presence or absence of 25 μM chloroquine (CQ), and the expression of LC3-I/II was measured by western blot analysis. One representative experiment is shown. β-actin was used as internal control. (d) Findings as in (c) were assessed by quantitative analysis of LC3II-I/β-actin protein levels and shown as histograms. The data are presented as the means ± S.D. from three independent experiments. *P < 0.001