Fig 2: Inhibition of SelK SH3 domain protects mice from NAFLD(A) Histopathological examination of AAV injected C57BL/6 J livers. Representative pictures of HE staining (above) and ORO staining (below) in liver sections from mice (n = 6). (B) Representative images of immunofluorescence staining of CD36 subcellular distribution in each group of mice livers (n = 5). Staining with antibodies against CD36 (green) and Calnexin (red, above), GM130 (red, middle) and Caveolin (red, below). (C) Histograms represent H&E and Oil Red O staining of liver tissues. (D) Quantification of the percentage of CD36 localized in ER-positive region (left), Golgi-positive region (middle) and Plasma Membrane-positive region (right), respectively. (E) Co-immunoprecipitations of Sec24 and CD36 in mice livers. (F) Expression of CD36 in plasma membrane and total proteins extracted from mice livers (n = 6). (G) Quantification of CD36-Sec24 combination level (left) and plasma membrane expression level (right) of CD36 in mice livers from the different groups. All data are shown as means ± s.d., *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig 3: SelK is involved in the fatty acid uptake activity of CD36(A) SelK is required for the plasma membrane localization of CD36. Representative confocal microscopy images of CD36 induced by overexpressed SelK or knockdown SelK in HepG2 cells. Fluorescence intensity of CD36 on plasma membrane (PM). Data are shown as means ± s.d., **P < 0.01, ***P < 0.001. (B) BODIPY staining in HepG2 cells transfected with SelK lentivirus (above) and shSelK lentivirus (below) (n = 5). Data are shown as means ± s.d., ***P < 0.001, ****P < 0.0001. (C) Knockdown of SelK reduced fatty acid (FA) transport via CD36. On Day 4, cells were incubated with FL-C16 for 30 min at 4 °C (above) and 37 °C (below) and labeled with PE-CD36, followed by a bivariate FACS analysis. Low-capability of long-chain fatty acid (LCFA) binding in SelK-knockdown-HepG2 cells (above). Low-capability of LCFA uptake in SelK-knockdown-HepG2 cells (below). Independently repeated for 5 times.

Fig 4: SelK increases the activation of Sar1B and triggers nascent COPII vesicles formation(A and B) On Day 0, HepG2 cells were infected with lentivirus expressing SelK or SelK-knockdown were set up as in Fig. 3. On Day 2, cells were harvested, Sar1B expression were analyzed by Western blot. (C) mRNA expression of Sar1B in HepG2 cells transduced with scrambled or SelK expressing lentivirus (left)/SelK shRNAs (right) as measured by qRT-PCR. (D) SelK prolongs the life-span of Sar1B. On Day 0, SelK overexpressing and knockdown HepG2 cells were set up as in (A). On Day 2, cells were treated with 100 mg/mL CHX for the indicated time. Cells were harvested, Sar1B degradation were analyzed by Western blot (n = 3). (E) SelK promotes Sec12 - induced activation of Sar1B. HepG2 cells were set up as in (A). Co-immunoprecipitations of Sec12 - Sar1B in HepG2 cells (n = 3). All data are shown as means ± s.d., *P < 0.05, **P < 0.01, ****P < 0.0001, nsP > 0.05.

Fig 5: SelK accelerates the cargo CD36 integration into COPII vesicles(A and B) HepG2 cells were infected with lentivirus expressing SelK or SelK-knockdown were set up as in Fig. 3. (A) Co-immunoprecipitations of CD36 - Sec24 in HepG2 cells (n = 3). (B) Dual-immunoflorescent staining of CD36 (green) and Sec24 (red) in cells (n = 5). (C–E) HepG2 cells were transfected with wild-type SelK lentivirus (SelK Full-length) or SelK lacking the SH3-binding domain lentivirus (SelK SH3BD TRUNC). (C) Cells were harvested and subjected to an Acyl-RAC assay (n = 3). Input and eluted fractions were separated on SDS gels and subjected to immunoblotting with anti-CD36 antibodies. (D) Cells were harvested and subjected to surface biotinylation. Equal volume of input and eluted fraction for each treatment was loaded on an SDS-PAGE gel and immunoblotted with β-actin antibody. (E) Deletion of the SH3 binding region in SelK decreased the endogenous CD36 that are enriched in the Golgi apparatus. On Day 0, HepG2 cells transduced with SelK full-length or SelK SH3BD truncated lentivirus were set up as described in (A). On Day 2, cells were incubated with serum-free medium for 12 h followed by 24 h of 160 μM PA treatments. On Day 3, cells were subjected to immunofluorescence assay using the anti-CD36 (green) and anti-GM130 (red, left) or anti-Calnexin (red, right) antibodies. (F) Quantification of CD36 palmitoylation level in cells. (G) Quantification of surface biotinylation results of the CD36 in cells. (H) Pearson's correlation of CD36 and Golgi apparatus (left) or ER (right). All data are shown as means ± s.d., *P < 0.05, ***P < 0.001, ****P < 0.0001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)