Fig 2: PNPLA8-mediated autophagy was regulated by mTOR inhibition.(a) 293T cells were transfected by the indicated plasmids. Following transfection, cells were cultured in medium with (10 μg/ml) or without insulin and harvested in 30 min. The cell lysates were prepared and total and phosphorylated forms of the indicated proteins were analyzed by western blot. β-actin was used as a sample loading control. (b,c) The densitometries for pmTOR and pUlk1 blots from a were compared as β-actin blot as a control (d) Quantitative analysis of p-Ulk1 positive stains in HEK293 cells expressing GFP as a control, WT, or Mut. Cells were fixed and stained with anti-pUlk1 (S757) antibody before being analyzed by confocal microscopy. Each bar indicating the percent of p-Ulk1 positive stains in WT expressing cells (open bars) or in Mut expressing cells (filled bars) is relative to p-Ulk1 positive of GFP expressing cells. Percentage of p-Ulk1 positive cells were calculated as follow: (p-Ulk1 in WT cells/pUlk1 in control cells) × 100 or (p-Ulk1 in Mut cells/pUlk1 in control cells) × 100.

Fig 3: Statin induces autophagy in primary hepatocytes and Pnpla8 is upregulated by SREBP-2.Mouse primary hepatocytes (MPH) were prepared from HFD treated mice and dishes were incubated with different concentrations of lovastatin for 24 hr and then analyzed. (a) Acidic vacuoles (AVOs) were measured using acridine orange (AO) staining. Cells were analyzed by flow cytometry and the percentage of AO positive cells was determined. (b) Dishes were treated with vehicle or 1 mM 3-MA for 1 h prior to AO staining and flow cytometric evaluation. The red arrow indicates AO staining was reduced from 25.12% to 14.65% by 3-MA. (c) Dishes were prepared for confocal imaging of AO staining acidic vacuoles (red) and live cells (green). (d) Cell extracts were analyzed by immunoblotting to measure the autophagy-related proteins Atg5, Atg7, and LC-3B. (e) A genome browser view of SREBP-2 ChIP-Seq read mapping data (Seo et al.12) (11) detected a peak around SREBP-2 consensus sequence within the Pnpla8 promoter (chr12:45370150-45370250). (f) Gene-specific manual ChIP analysis in hepatic chromatin from HFD + L/E-treated group was performed using an antibody against SREBP-2 (filled bars) or control IgG (open bars). Gene-specific primers for known SREBP binding peaks in the indicated promoter regions were used in the qPCR. Data are shown as means ± standard deviations. *p < 0.05. All data are representative of three independent experiments. (g) RNA harvested from mouse primary hepatocytes from HFD treated mice cultured for 24hr in the presence of sterols (open bars) or the absence of sterols plus 5 uM lovastatin for 24 hr. (filled bars) was subjected to Pnpla gene expression profiling via q-PCR. Target gene expression levels were normalized to levels of the housekeeping gene Rplpo using the ΔCt method. (h) MPH from HFD treated mice were cultured for 24 hr with media containing sterols or sterol depleted media containing 5 uM lovastatin and PNPLA8 protein were measured.

Fig 4: Tracking of the functional properties of Pnpla8 in vivo. Mice were sacrificed 12 days after transfection with control GFP (a,d) or PNPLA8-GFP expressing plasmids (b,e). Hematoxylin and eosin (H&E)-staining (a,b) or con-focal GFP and Bodipy imaging (d,e) were performed. Triglyceride (TG) were measured from the tissues lysates (c). Student’s t-tests were used for the statistical analysis. *p < 0.05.

Fig 5: PNPLA8 expression correlates with lipid droplet depletion in primary hepatocytes.MPH from HFD treated mice were transfected with a control or PNPLA8 siRNA for 24 hr and then cultured with medium supplemented with lipid-depleted media and lovastatin (final 5 uM) for the indicated times. (a) Triglyceride levels (TG) were measured in total cell lysates and plotted as a relative level of the starting value which was 154 mg/g protein. (b) Pnpla8 protein levels were measured as indicated time points. (c) The MPH were cultured with ( + ) or without (−) statin as shown in a and evaluated for the localization of lipid droplets (BODIPY 493/503, red and PNPLA8 (green). (d) Whole cell lysates were analyzed for autophagy related-proteins via immunoblotting. (e,f) Immunoblots for autophagy-related proteins from primary hepatocytes cultured as above and treated for 24 hr with si-control or si-PNPLA8. (g) The AO positive cells with (5 uM) or without lovastatin were compared to siSREBP2 –treated dish. All data are representative of three independent experiments. Data are shown as means ± standard deviations. *p < 0.05.