Figure 1

CAV1 knockdown impedes autophagy activation by H₂O₂. (a and b) Control (shCtrl.) and CAV1 knockdown (shCAV1) HeLa stable cells were transfected with mCherry-GFP-LC3 for 24 h and then treated with 3 mM H₂O₂ for 30 min. Cells were fixed with 4% paraformaldehyde for 5 min and then observed under a confocal microscope (scale bars=10 μm) (a). The number of yellow (GFP and mCherry-double positive) or red puncta per cell in confocal images were quantified (mean values±S.D., n=10; ^#P<0.05, ^##P<0.005 versus veh. shCtrl.; *P<0.05, **P<0.005 versus H₂O₂ shCtrl.; NS, not significant versus veh. shCtrl.) (b). (c and d) Control (shCtrl.) and CAV1 knockdown (shCAV1) HeLa stable cells were treated with 3 mM H₂O₂ for 30 min and analyzed by western blotting (c). The signals on the blot were quantified using Science Lab software (Hercules, CA, USA) and the relative ratios of LC3-II to TUBA were calculated (mean values±S.D., n=3; ^**P<0.005 versus shCtrl. cells) (d). (e) CAV1-WT and CAV1-KO HeLa cells were treated with 3 mM H₂O₂ for 30 min, and subjected to electron microscopic analysis. Autolysosomes (ALs, asterisks), mitochondria (M), autophagosomes (APs), golgi apparatus (Golgi) and nuclei (N) are indicated. (f) The numbers of cytoplasmic ALs and APs were counted (mean values±S.D., n=10; ^#P<0.05, ^##P<0.005 versus veh. CAV1-WT cells; *P<0.05, **P<0.005 versus H₂O₂-treated CAV1-WT cells; NS, not significant versus veh. CAV1-WT cells)