Fig. 5

DIRAS3 and p53 re-expression stimulates autophagy in HNSCC cells. a. CAL-27 cells were treated with Ad-DIRAS3, rAd-p53, and their combination for 24 h. Ad-GFP was used as a negative control. Autophagy was determined by examining the cellular AVs using transmission electron microscopy. Representative images of cells that developed AVs are shown. White arrows indicate AVs. Scale bar, 5 μm. b. The formation of GFP-LC3 puncta was observed using fluorescence microscope. The percentage of cells with GFP-LC3 puncta in each group was as follow: Control group: 4.67% ± 1.25%, DIRAS3 group: 40.33% ± 5.73%, p53 group: 34.00% ± 2.94%, DIRAS3 plus p53 group: 50.00% ± 4.55%. Representative images and the quantification of GFP-LC3 puncta are shown. Scale bar, 20 μm. c. CAL-27 cells were treated with Ad-GFP, Ad-DIRAS3, rAd-p53, and Ad-DIRAS3 plus rAd-p53. After 24 h of infection, cells were further starved in EBSS or treated with 100 nM Baf A1 for 3 h or 6 h. The formation of GFP-LC3 puncta was observed using fluorescence microscope. Representative images and the quantification of GFP-LC3 puncta are shown. The autophagic flux was deduced from the ratio between the percentage of cells with GFP-LC3 puncta in the presence of Baf A1 and the percentage in the presence of PBS [17]. Scale bar, 20 μm. d. CAL-27 cells were treated as in (c). The levels of LC3-I and LC3-II were detected by western blotting. The expression of LC3-II was calculated after normalizing to GAPDH. The autophagic flux was deduced from the LC3-II expression levels in the presence of Baf A1 compared to the levels in the presence of PBS [17]. *P < 0.05; **P < 0.01; ***P < 0.001