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. 2019 Sep 4;16(6):1111–1129. doi: 10.1080/15548627.2019.1659613

Figure 6.

Figure 6.

LC3B interacts with the CSD in CAV1 to inhibit autophagy, but interacts with the IMD in CAV1 to activate autophagy. (A) Schematic representation of the plasmids carrying Flag-CAV1 or Flag-CAV1 mutants, CAV1: CAV1 wild type; LIRs−/-(CSD): CAV1 mutant carrying the defective LIRs in the CSD; LIRs−/-(IMD): CAV1 mutant carrying the defective LIRs in the IMD; LIRs−/-: CAV1 mutant carrying the defective LIRs in both the CSD and IMD. (B) HUVECs were transfected with plasmids encoding Flag-CAV1 wild type (0.4 μg) or Flag-CAV1 mutants (0.4 μg) respectively, and co-transfected with GFP-LC3B plasmids for 24 h, followed by 30 mM glucose (glu) treatment for 24 h. GFP-LC3B puncta was visualized by confocal microscopy. Representative fluorescent images are shown (upper panel). Scale bars: 15 μm. The number of GFP- LC3B puncta in each cell was quantified, and at least 50 cells were included for each group (lower panel). * p < 0.05 versus CAV1 + 5.5 mM glucose; † p < 0.05 versus LIRs−/- (CSD) + 5.5 mM glucose; # p < 0.05 versus LIRs−/- (IMD) + 5.5 mM glucose (n = 3). (C-F) HUVECs were transfected with plasmids encoding Flag-CAV1 wild type (0.4 μg) or Flag-CAV1 mutant (0.4 μg) for 24 h, and treated with high glucose (30 mM, 24 h). Whole-cell lysates from HUVECs were subjected to immunoblot analysis to determine Flag-CAV1, LC3B, and SQSTM1 levels. glu: glucose Representative western blotting analysis of the indicated proteins (C) and summary bar graph showing the expression of the indicated proteins (D). n = 3. * p < 0.05 versus CAV1; † p < 0.05 versus LIRs−/- (CSD); # p < 0.05 versus LIRs−/- (IMD). (E-F) Whole-cell lysates from HUVECs treated as above mentioned, and then immunoprecipitated with Flag antibody (E; IP: Flag) or LC3B antibody (F; IP: LC3B). Cellular ACTB derived from the same samples for IP experiments served as a loading control. glu: glucose. (G) HUVECs were transfected with plasmids encoding, vector or GFP-CAV1 (0.4 μg) or GFP-CAV1 mutants (0.4 μg) respectively, and then were analyzed by immuno-electron microscopy using primary antibody against GFP and 4 nm colloidal gold affinipure goat anti-mouse IgG as secondary antibody. Arrow, autophagosomes or autolysosomes; Asterisk, 4 nm colloidal gold in autophagosomes or autolysosomes; scale bar: 200 nm.