Fig 2: Activation of the p62-mediated noncanonical KEAP1-Nrf2 pathway is dependent on endoplasmic reticulum (ER) stress and protects cells against lipotoxicity. Hepa1c1c7 cells were treated with palmitic acid (PA; 500 μM) and 4-phenylbutyric acid (4-PBA; 1 mM) for 18 h. (A) Immunoblot analysis using antibodies against p-p62 (S351), p62, KEAP1, nuclear Nrf2, LaminB (nucler protein marker), and ACTB (loading control). (B, C) Densitometric analysis of the immunoblots. Quantitative RT-PCR (qRT-PCR) analysis of Keap1 (D), Gsta1 (E), HO-1 (F), and Nqo1 (G) mRNA. (H–I) Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) analysis of cells treated as described in (A). Scale bar: 100 μm. (I) Quantitative analysis of TUNEL-positive cells. (J) Cell viability was evaluated using a Cell Titer-Glo assay kit. The number of live cells have expression as absorbance at luminescence. (K) The levels of reactive oxygen species (ROS) were determined using CM-H2DCFH-DA. Scale bar: 100 μm. (L) Quantitative analysis of relative DCF fluorescence. (M) Immunoblot analysis of cells that were treated as described in (A) using antibodies against Grp78, p-PERK, PERK, cleaved ATF6 (C.ATF6), p-IRE1α, IRE1α, and ACTB (loading control). (N–O) Densitometric analysis of the immunoblots. qRT-PCR analysis of Trb3 (P), PERK (Q), Grp78 (R), and Atf4 (S) mRNA. Graphical and statistical analyses were performed using GraphPad Prism 9 software. Bar graphs were drawn using the mean values of the results per sample but the statistical significances were derived from raw data. Data are presented as means ± SD from 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, and N.S., not significant. See also Fig. S1.

Fig 3: Fas interacts with p62 and colocalized with LC3B in Cd-exposed Raw264.7 cells. (A, B) Cells were treated with 30 µM Cd for 6 and 12 h, respectively. Lysates were immunoblotted for p62, and the other aliquot (600 µg) were immuniprecipitated with Fas antibody (antibody clone G-9) and mouse IgG, followed by immunoblotting for p62. (C, D) Cells were treated as described in above. Lysates were immunoblotted for Fas (clone G-9), and the other aliquot (600 µg) were immuniprecipitated with p62 antibody and mouse IgG, followed by immunoblotting for Fas (clone G-9). All data shown are presentative of over three separate experiments. H, Ig indicates immunoglobulin heavy chain. GAPDH was used as the loading control. (E) Cells cultured on coverslips were exposed with Cd (30 µM) for 12 h prior to fixation and stained with LC3B (green) and Fas (red) antibodies, and FITC and rhodamin as the secondary antibody, respectively. Nuclei were counterstained with Hoechst 33342 (blue) and photographed. Cells were imaged using a 40X objective. The scale bar is 25 µm.

Fig 4: Decreased mTOR activation in p62 KO mice after APAP treatment. Male WT and P62 KO mice were treated with APAP (500 mg/kg, i.p.) or saline for 24 h. (A) Western blot analysis of S6, p-S6, 4EBP1, and p-4EBP1 of total liver lysates. (B) Densitometry analysis of p-S6 and p-4EBP1 from (A). Data are presented as mean ± SEM (n ≥ 3). (C) Representative images of the immunohistochemical staining of the liver p-S6 in the indicated mice (magnification 200×).

Fig 5: High phosphate caused cardiac remodeling and dysfunction and low autophagy activity in the hearts of WT mice. WT mice (A–D) or GFP-LC3 reporter mice (E,F) were fed normal- (N Pi) or high-phosphate (H Pi) diet for 12 weeks. After 12 weeks, mice were subjected to cardiac MRI for evaluation of cardiac function (A) and morphometry (B) followed by euthanization for heart histology study (C) and immunoblots (D). (A) Left ventricular ejection fraction; (B) left ventricular wall thickness at systole (left panel) and diastole (right panel); (C) heart hypertrophy in mice fed with high dietary phosphate. Left panel: representative gross appearance of heart. Right panel: heart weight/body weight. (D) Changes in autophagic markers in the heart. Left panel: representative immunoblots for LC3 and p62 in left ventricular lysates. Right panel: a summary of all immunoblot data. (E) Autophagic activity in the hearts of GFP-LC3 reporter mice. Four hours prior to sacrifice, mice were treated intraperitoneally with chloroquine (50 mg/Kg body weight). Left panel: representative micrographs of GFP-LC3 immunofluorescence puncta in free walls of left ventricles. Right panel: semi-quantitation of GFP-LC3 puncta. Sample number in each group is in brackets underneath the corresponding bars. Quantitative data are presented as mean ± SD with scatter plots of individual data points, and statistical significance was assessed by unpaired Student’s t-test. Significant differences are accepted when * p < 0.05 or ** p < 0.01 between two groups. (F) Correlation between cardiomyocyte size and the number of LC3 puncta in the hearts of LC3 reporter mice treated with normal (black symbols) or high (red symbols) dietary phosphate. The number of LC3 puncta in one heart section was counted in three locations (up, middle, and bottom) of the free wall of left ventricle; therefore, 6 mice gave 18 datasets in either the normal- or high-phosphate-fed group.