Fig 2: HCV infection induced the co-localization of ApoJ with SOAT2 and elevated SOAT activity in the context of Golgi dispersion.a Protein levels of SOAT1 and SOAT2 in Huh7.5 cells with acute or chronic HCV infections, with the intensities shown in the right panel (n ? 2). b HCV-infected Huh7.5 cells were fed with NBD-Chol, and the SOAT activity was evaluated (n = 6). c HCV-infected Huh7.5 cells were treated with 0.25–2 µM TMP153 for 16 h and the SOAT activity was quantified as in b. The asterisk indicates statistical significance (p < 0.05) as compared to the untreated control. d Representative IFA images of SOAT2 (red) and Golgi (TGN38, green) in HCV-infected cells. The scatter plots of red and green channels, as shown at the right of the merged image, and the Pearson’s and Mander’s colocalization coefficients were analysed by the Coloc2 plugin from ImageJ/Fiji (ImageJ-Fiji-ImgLib http://fiji.sc/), respectively, as shown in the right panel. The fluorescence intensity profile across the arrow for both channels was analysed by FVW31S software. e Representative IFA images of SOAT2 (red) and ApoJ (green) analysed as in d. Scale bar, 5 µm.

Fig 3: FFA-induced hepatic lipid accumulation depended on ApoJ.The cellular contents of TC and TG were quantified in Huh7 cells treated with PA (a) or OA (b) for 24 h (n ? 5), CE/FC ratio of PA and OA treated cells (c) (n ? 6), and ApoJ expression in Huh7 cells treated with PA (d) or OA (e). The lower panel: ApoJ intensities (n = 3); psApoJ, ApoJ precursor. f Representative images of LDs stained with BODIPY493/503, and g quantification of lipid contents in Huh7.5 cells bearing Luc or ApoJ shRNA treated with PA or OA for 24 h. Scale bar, 100 µm in f. h Quantification of lipid contents in the cells transfected with ApoJ expressing plasmid or control vector. All results are normalized to the respective untreated control and presented as the mean ± SEM (n ? 5), and asterisks indicate statistical significance (p < 0.05). Representative IFA images of SOAT2 (red) and Golgi (TGN38, green) (i); SOAT2 and ApoJ (green) (j) in PA- or OA-treated cells were analysed by Coloc2 plugin and FVW31S software as in Fig. 2d. Scale bar, 5 µm. Asterisks indicate statistical significance (p < 0.05). k The PA- (200 µM) or OA- (800 µM) treated Huh7 cells were fed with NBD-Chol, and the SOAT activity was evaluated and expressed as the mean ± SEM (n = 6). l The protein level of SOAT2 in Huh7 cells treated with PA or OA for 24 h. The lower panel: SOAT2 intensities (n ? 2). m Huh7 cells were treated with 800 µM OA for 24 h. The cell lysate was immunoprecipitated with antibody against ApoJ and examined by western blot analysis recognizing SOAT1, SOAT2, and ApoJ.

Fig 4: ApoJ silencing reduced SOAT2 expression and SOAT enzymatic activity.Huh7.5 cells bearing Luc or ApoJ shRNA were established to examine a SOAT2 mRNA (n = 3); b SOAT2 protein expression (representative image in left panel; SOAT2 intensity in right panel, n = 4) and; c SOAT enzymatic activity in the presence of FFAs or HCV infection at MOI = 0.01 for 6 days with and without TMP153 treatment; and d, e image visualization of NBD-CE under confocal microscopy observation. The data were presented as the mean ± SEM (n ? 6), and asterisks indicate statistical significance (p < 0.05) between the control and ApoJ knockdown cells. Scale bar, 5 µm.