Fig. 3: PNO1 inhibits cell apoptosis by promoting autophagy.

A Autophagy was evaluated using TEM in Hep3B sh-Ctrl and sh-PNO1 cells. The arrows indicate autophagosomes or autolysosomes. Scale bar = 5 μm (upper) or Scale bar = 1 μm (lower). B Hep3B sh-Ctrl, Hep3B sh-PNO1, HLE Vector, and HLE PNO1 cells were immunostained with antibodies against LC3B. Scale bar: 50 μm. C Western blotting analysis of autophagy-related protein level. D, E Quantification of relative protein expression. F–H Hep3B sh-Ctrl, Hep3B sh-PNO1, HLE Vector, and HLE PNO1 cells transfected with GFP-mRFP-LC3 lentivirus. Hep3B sh-PNO1 and HLE PNO1 cells were pretreated with rapamycin and 3-MA, respectively. Images were then acquired by fluorescence microscopy (F) and the average number of yellow dots (autophagosomes) or red-only dots (autolysosomes) in the merged images per cell was quantified (G, H). Scale bar: 5 μm. I–K Apoptotic rate was measured using annexin V/PI double staining in cells as in (F). L, M CCK-8 assay assessed the viability of cells as in (F). N Western blotting analysis of apoptosis and autophagy-related protein levels in cells as in (F). O, P Quantification of relative protein expression. Data were presented as mean ± SEM. n = 3–4. *p < 0.05, **p < 0.01, ***p < 0.001.