Fig 2: Loss of TSPO caused autophagy owing to FC accumulation(A) Representative electron micrographs showing the ultrastructure of Scram and TSPO KD cells. Mitochondria (red arrow), autophagosome (black arrow) in TSPO KD after AcLDL treatment and autophagolysosome formation (double black arrows) in TSPO KD after AcLDL+58035 treatment as compared with the corresponding control. Mit, mitochondria; ER, endoplasmic reticulum (black arrowhead); LD, lipid droplet; Pero, peroxisome. Scale, 200 nm.(B) Immunoblot of LC3 (upper) and LAMP1 (lower) in Scram and TSPO KD cells treated with DMSO, AcLDL, and AcLDL+58035. The ratio indicates the densitometric analysis of LC3-II/GAPDH.(C and D) Immunoblot of LC3 (C), ATG12 and LAMP2 (D) in WT, and TSPO KO mouse hepatocytes treated with DMSO, AcLDL, and AcLDL+58035. LC3-II was quantified with GAPDH in (C). The ratio indicates the densitometric analysis of LC3-II/GAPDH, ATG12/GAPDH, and LAMP2/GAPDH. Data are represented as mean ± SD, *p < 0.05, **p < 0.01 by one-way ANOVA.(E) Immunoblot of LC3 in Huh7 cells treated with DMSO, AcLDL, and AcLDL+19-Atriol.

Fig 3: TSPO is an indicator of the progression of NAFLD(A) Immunohistochemistry staining showing TSPO levels in human liver paraffin sections (n = 3). The brown color indicates a positive TSPO signal (black arrow, left panel). After the removal of areas of blood and vessels by ImageJ software, quantification showed the TSPO positive signal in each field in normal, steatosis, NASH, and cirrhosis liver (right panel). Scale, 200µm.(B) Immunofluorescence staining showing TSPO immunoreactivity signal in control (chow diet), steatosis, and NASH mouse models. The upper panels are low magnification, and the lower panels are high magnification of the selected dotted areas. Red colors indicate positive TSPO signal (white arrow). TSPO signals were quantified in control, steatosis, and NASH models. Scale, 40 µm.(C) qPCR data showing Tspo/Gapdh relative mRNA expression in mouse liver of control, steatosis, and NASH (n = 3). Data are represented as mean ± SD, *p < 0.05, **p < 0.01 by one-way ANOVA.

Fig 4: Total cholesterol or free cholesterol and TAG accumulation in TSPO-deficient cells(A) Immunoblotting data showing Huh7 TSPO knock down (KD) cells. ß-actin is a loading control.(B) Colocalization of TSPO (green) and MitoTracker (Mito; red) under confocal microscopy. Scale, 10µm.(C) MTT assay for Scram and KD cells treated with DMSO, AcLDL, or AcLDL+58035 for 24 h.(D) Determination of TC, FC, and CE content in Scram and TSPO KD Huh7 after treatment with DMSO, AcLDL, and AcLDL+58035 for 24h.(E) Determination of TC, FC, CE content in WT and TSPO KO primary mouse hepatocytes after treatment with DMSO, AcLDL, and AcLDL+58035 for 24 h.(F) Determination of TAG in Scram and KD cells after treatment with DMSO, AcLDL, and AcLDL+58035 for 24 h.(G) Determination of TC, FC, and CE after treatment with DMSO, AcLDL, and AcLDL+19-Atriol for 24 h in Huh7.(H) MTT assay for Huh7 cells treated with DMSO, AcLDL, and AcLDL+19-Atriol for 24 h.(I) Measurement of TC in WT and TSPO KO mouse liver by MALDI IMS. Upper: coregistration of MS image with the optical image showing morphological features: The green indicates TC perfusion throughout the liver tissue in the hepatic lobules and around vasculature; the red indicates the distribution of heme molecule from blood cells revealing vasculature. Lower: intensity plots showing cholesterol distribution in the WT and TSPO KO liver. Scale, 5 mm.(J) Measurement of TAG (50:2) content in the WT and TSPO KO mouse liver by MALDI. Upper: the yellow indicates TAG tissue distribution and the blue shows absence of TAG from respective tissue compartments (blood vessels or biliary ducts). The TAG signal is illustrated as a color gradient from dark blue which represents absence of TAG to yellow which shows strong TAG signal. Lower: intensity plots showing TAG distribution in WT and TSPO KO liver. Scale, 5 mm. Data are represented as mean ± SD, *p < 0.05, **p < 0.01, ***P < 0.001 by one-way ANOVA.

Fig 5: Loss of TSPO inhibited ACAT2 independent of cholesterol accumulation and TSPO-ACAT2 interacted in MAMs(A) CEH immunoblots from DMSO-, AcLDL-, and AcLDL+58035-treated Scram and TSPO KD Huh7 cells. The numbers under the lanes represent the CEH/GAPDH.(B) ACAT2 immunoblot from DMSO-, AcLDL-, and AcLDL+58035-treated Scram and TSPO KD Huh7 cells. The bar graph is the fold change of ACAT2/GAPDH.(C) Immunofluorescence staining of ACAT2 (green) in DMSO-, AcLDL-, and AcLDL+58035-treated Scram and TSPO KD Huh7 cells. Scale, 10 µm.(D) CEH immunoblot from DMSO-, AcLDL-, and AcLDL+58035-treated WT and TSPO KO primary mouse hepatocytes. The numbers under the lanes represent the CEH/GAPDH.(E) ACAT2 immunoblot from DMSO-, AcLDL-, and AcLDL+58035-treated WT, TSPO KO primary mouse hepatocytes. The bar graph is the fold change of ACAT2/GAPDH.(F) Immunofluorescence staining of ACAT2 (green) in DMSO-, AcLDL-, and AcLDL+58035-treated WT and TSPO KO primary mouse hepatocytes Scale, 10µm.(G) Coimmunoprecipitation of ACAT2-Turbo-GFP and TSPO-Myc and immunoblot with Anti-GFP or Anti-C-Myc in Huh7 cells.(H) PLA of ACAT2 and TSPO interactions (red spot) in Huh7 cells. Scale, 10 µm. Data are represented as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001 by Student's t-test.