Figure 2.

HNG stimulates lysosomal protein degradation but not macroautophagy. (A) Long-lived protein degradation in NIH3T3 cells treated with HNG, PQ alone, and combination of PQ and HNG. n = 6. (B and C) Effect of HNG on LC3-II turnover in NIH3T3 (B) and H9C2 (C) cells treated or untreated with protease inhibitors (PIs). Left: Representative immunoblots. Right: Quantifications of changes in LC3-II normalized for actin. n = 3–5. (D and E) Effect of HNG on autophagic flux measured in NIH3T3 (D) and H9C2 (E) transduced with a lentivirus expressing the tandem reporter mCherry-GFP-LC3 and cultured the presence of the indicated concentrations of HNG alone (none), with PQ (10 mM), or in the absence of serum. Left: Representative images. Insets show higher-magnification images. Right: Quantification of changes in the mean number of autophagosomes (APGs; yellow puncta) or autophagolysosomes (APGLs; red puncta only) per cell section quantified with high-content microscopy in n = 1,200 cells per condition. Bars, 10 µm. Values are means + SEM. Please note that SEM in D and E are not visible because they were <0.1% of the total value. Differences with untreated cells are significant for *, P < 0.05.