Fig. 2: Regulation of autophagy signal by VHL expression.

a The 786-o or 786- HA-VHL cells were cultured in complete media with 10% FBS or serum-free media for 24 h and analyzed using western blotting. b The 786-o or 786-HA-VHL cells were transfected with 10 µg GFP-tagged LC3B plasmid, cultured under the same conditions as in Fig. 2a, and observed using a fluorescence microscope. c The GFP-LC3B puncta per cell (N = 10/group) were quantified using the ImageJ software. d HeLa cells were transfected with 15 µg scrambled shRNA or 5 and 15 µg VHL-shRNA plasmid. After transfection for 48 h, the cells were analyzed using western blotting with the indicated antibodies. e After transfection of HeLa cells stably expressing GFP-LC3B with scrambled shRNA or VHL-shRNA plasmids, the cells were cultured in complete media (10% FBS) or serum-free media (0% FBS) with or without 10 µM bafilomycin A1 treatment for 24 h. Subsequently, the cells were observed using fluorescence microscopy. f The puncta in the representative image were quantified using the ImageJ software. g HeLa cells stably expressing GFP-LC3B were transfected with scrambled shRNA or VHL-shRNA plasmids, stained with anti-LAMP1 (used as a lysosome marker), and observed using confocal microscopy. DAPI was used for nuclear staining. h The representative image was quantified using the ImageJ software. Error bars indicate the S.D. of means (*P-value < 0.05, **P-value < 0.01). i The Atg5 knockout MEFs were either left untreated or were treated with 20 ng/ml doxycycline hydrochloride (DOX) for 5 days. The treated/not-treated Atg5 KO MEFs were transfected with 15 µg Flag-VHL plasmid, cultured in complete medium with 10% FBS or serum-free DMEM for 24 h, and then analyzed using western blotting