Fig 2: Increased expression of autophagic proteins in PSC.Western blotting of p62, ATG5/12, ATG7, Beclin1, LC3B, LAMP1 and LAMP2 in liver extracts prepared from patients with PSC and Controls. Blots were normalized to GAPDH expression. Data are presented as Mean ± STDEV and were statistically analyzed using Student’s t-test, *p<0.05, **p<0.01, (N = 6/group).

Fig 3: NLRX1-mediated incomplete mitophagy in morphine-treated BV2 cells. A Confocal microscopy analysis of MitoTracker Green Tm and LysoTracker Red DND-99 in BV2 cells of NC-siRNA or NLRX1-siRNA group at the absent or present of morphine. Bar = 5 µm. Quantitative graphs of mitochondrial form factors (B), average sizes (C) and lysosomal diameters (D) as measured by Image J (n > 25 cells). E–F Representative images of confocal microscopy analysis of LAMP1 and LC3B and quantitative analysis of overlap coefficients, lysosomal phagocytosis ratio of LC3B and LC3 + phagosomes (F, n = 24–36 cells). Bar = 2 µm. Data represent the mean ± SEM. Student's t-test or Mann–Whitney U test were used to measure significance between two groups. (* p < 0.05, ** p < 0.01, *** p < 0.001 and ns p > 0.05)

Fig 4: Increased accumulation of p62, LC3b, LAMP1 and LAMP2 in PSC and BDL mice is located primarily in parenchymal cells that surround the portal triad.Tissue sections from normal human liver, PSC, sham and BDL mice were examined for p62, LC3b, LAMP1 and LAMP2 expression. A. Human control and PSC. B. Sham and BDL. N = 3-5/group, 200X, CV-central vein, PT-portal triad. Arrows indicate increased staining. C. 10-week old WT and Mdr2KO (200X). N = 3-5/group, 200X, CV-central vein, PT-portal triad. Arrows indicate increased staining.

Fig 5: Unresolved excess cholesterol deposition and inflammation in spinal cord lesions. (A) Representative images of spinal cord lesions stained with LAMP1 (green), MAC2 (red), and DAPI (blue) at different time points after injury. (B) Immunoblots of spinal cord lesions for ABCA1, CTSD, caspase-1, IL-1ß, and ß-actin (loading control) at different time points after injury. (C) Representative images of spinal cord lesions in different groups stained with ORO (red) taken at 4 wpi. (D) Quantification of the ORO-positive area in (D) (n = 3, 4, and 3 mice for Control, GW3965 and APOE KO, respectively). (E) Reflection images of lesion cores in different groups showing crystals (white) taken at 4 wpi. (F) Quantification of the crystal intensity (normalized to the proximal region) in (F) (n = 3 mice). (D and F) Ordinary 1-way ANOVA with Tukey’s multiple comparisons test. All data are shown as mean ± SEM. ns, not significant. Scale bars: 20 µm (A and E) and 200 µm (C).