Fig 2: Metformin promotes autophagy in FFA-treated human keratinocyte. HaCat cells were treated with 400 µM FFA for 10 days, and then the intervention groups were co-treated with metformin (40mM) and FFA for additional 4 days. (A) Immunoblot of LC3 and P62 in HaCaT cells. (B) Representative confocal images of HaCat cells expressing GFP-RFP- LC3 and quantitation of early autophagosome puncta and autolysosome puncta following FFA and metformin treatment. Yellow showed co-localization of GFP and RFP, indicating early autophagosomes. Red only showed autolysosomes, scale: 20 µm. Data are presented as the mean ± SD and are representative of three independent experiments. *p < 0.05.

Fig 3: Foam cells are characterized by autophagy deficiency and lysosomal dysfunction. A-C, Protein levels of LC3I/II and p62 were determined by Western blotting. Representative Western blotting A, and quantitative analysis B and C, displayed a reduced protein level of LC3II and increased protein level of p62. D, The mRNA expression levels of autophagic genes were determined by RT-PCR. E, Foam cells displayed reduced autolysosomes as shown by the reduced red puncta of mRFP-GFP-LC3. F-H, Measurement of lysosomal biogenesis and function. F, LysoTracker staining for lysosomal biogenesis. G, Protein level of CTSD for lysosomal degradation capability. H, LysoSensor staining for pH. *P < .05; **P < .01. Results are means ± SD of three independent experiments. The value represents fold of vehicle

Fig 4: Caspase-6-mediated N-terminal p62 cleavage fragment negatively regulates p62 droplets associated autophagosome formation.A Caspase-6-mediated N-terminal p62 cleavage fragment reduced LC3-positive puncta formation. Cells transfected with mCherry empty vector (mCh-vec) or mCherry-p62-N (mCh-p62-N) were stained with anti-LC3 and p62 (C-terminus) antibody (guinea pig) for endogenous LC3 and p62 proteins. Note that anti-p62 C-terminus antibody does not react with mCh-p62-N. Confocal images were acquired. Bar: 10 µm. Total LC3 puncta in each cell or p62 droplets associated LC3 puncta in each cell were quantified. The diameter and the count of puncta in each cell were assessed by ImageJ. For the size of LC3 puncta, each point in the plot represents the average size of LC3 puncta in each cell. n = 50 cells from three independently plated wells. More than 10,000 LC3 speckles were analyzed. Data are shown as mean ± sem. Statistical analysis was performed by Two-way ANOVA with Bonferroni post-tests. The F/degree of freedom/post hoc P values are indicated in each plot. *P < 0.05; ***P < 0.0001. B Caspase-6-mediated N-terminal p62 cleavage fragment reduced ATG16L1-positive puncta formation. Cells transfected with mCherry empty vector (mCh-vec) or mCherry-p62-N (mCh-p62-N) were stained with anti-ATG16L1 and anti-p62 (C-terminus) antibody (guinea pig) for endogenous ATG16L1 and p62 proteins. Note that anti-p62 C-terminus antibody does not react with mCh-p62-N. Confocal images were acquired. Bar: 10 µm. Total ATG16L1 puncta in each cell or p62 droplets associated ATG16L1 puncta in each cell were quantified. The diameter and the count of puncta in each cell were assessed by ImageJ. For the size of ATG16L1 puncta, each point in the plot represents the average size of ATG16L1 puncta in each cell. n = 50 cells from three independently plated wells. Data are shown as mean ± sem. Statistical analysis was performed by Two-way ANOVA with Bonferroni post-tests. The F/degree of freedom/post hoc P values are indicated in each plot. *P < 0.05; **P < 0.01; ***P < 0.0001. C Caspase-6-mediated N-terminal p62 cleavage fragment enhanced the levels of puromycin-induced ubiquitin puncta. HeLa cells were transfected with mCherry empty vector /HA-ubiquitin or mCherry-p62-N/HA-ubiquitin (HA-ub). After 20 h, cells were treated with vehicle or puromycin (5 µg/ml) for 5 h. Cells were stained with anti-HA and p62 (C-terminus) (guinea pig) antibody. Note that anti-p62 C-terminus antibody does not react with mCh-p62-N. Confocal images were acquired. Bar: 10 µm. The diameter and the count of puncta in each cell were quantified by ImageJ. n = 50 cells from three independently plated wells. Data are shown as mean ± sem. Statistical analysis was performed by one-way ANOVA with Bonferroni multiple comparison test. The F/degree of freedom/post hoc P values are indicated in each plot. ns not significant; **P < 0.01; ***P < 0.0001.

Fig 5: Cytotoxicity-modulated p62-droplet formation does not correspond to full-length p62 levels.A–C p62-droplet formation in cells treated with an array of cytotoxicity. HeLa cells were treated with vehicle (DMSO) for 4 h, TNFa (10 ng/ml) + CHX (50 µg/ml) (T + C) for 3 h, staurosporine (STS) (1 µM), MG132 (5 µM), puromycin (Puro) (5 µg/ml), PI-103 (1 µM) or Etoposide (Etop) (50 µM) for 4 h, respectively. A The cells were stained with anti-p62 antibody (produced in guinea pig). Confocal images were acquired with confocal microscopy. Bar: 10 µm. The diameter of the biggest p62 puncta (µm) in each cell was measured (ImageJ), and the number of p62 puncta > 0.5 µm in each cell was assessed (ImageJ). n = the number of cells, as shown in each plot. B Full-length p62 levels in the cells under cytotoxicity. The cells were lysed and subjected to immunoblot with anti-p62 and GAPDH antibodies, successively. Data were quantified with ImageJ. n = independent immunoblots. C Cell viability under the array of cytotoxicity. The cells were subjected to viability assays with the CellTiter-Glo Luminescent Cell Viability Assay kit (Promega). n = independently plated wells. Data are shown as mean ± sem (A). Statistical analysis was performed by one-way ANOVA with Dunnett’s multiple comparison test. The F/degree of freedom/post hoc P values is indicated in each plot. ns not significant; *P < 0.05; **P < 0.01; ***P < 0.0001.