Fig 2: Inflammatory activation results in the high GPAT3 expression.A GO analysis of differentially expressed genes (DEGs) in the normal and LPS-stimulated KCs (stimulated with 1 µg/ml LPS for 24 h). DEGs were classified under six categories as indicated. Red arrow indicates lipid metabolism as the top-ranking affected metabolism pathway (n = 3). B Heatmap showing the expression of lipid metabolism pathway genes, as measured using transcriptomics, in the normal and LPS-stimulated KCs (n = 3). C Volcanic map showing the GPAT3 was the most significant difference among lipid metabolism genes after LPS stimulated KCs for 24 h (n = 3). D qPCR and Western blot analysis of GPAT3 expression in normal and LPS (100 ng/ml)-stimulated KCs at different time points (n = 3). E Immunofluorescence staining for GPAT3 expression in KCs from the LPS-treated liver mice tissues. F4/80 was used as a KCs marker. DAPI was used to visualize nuclei. Scale bars represent 50 µm (n = 3). F The mRNA expression of GPAT3 in human liver from the healthy and NASH (7 healthy human and 9 NASH patients, Database were obtained from GEO database: http://www.ncbi.nlm.nih.gov/geo/, Data set number is GSE63067). G GPAT3 mRNA expression in KCs after stimulated with TNF-a (100 ng/ml) for 24 h (n = 3). H LPA concentrations in the supernatant of KCs after stimulated with LPS for 24 h (n = 6). I Lipidomic analysis showing the levels of different LPC species levels in KCs (n = 6). Data represents mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 3: GPAT3 promotes inflammation response through the LPA-mediated PKC?, ERK1/2 and c-Jun pathway.A Mitochondrial membrane potential (JC-1) in KCs treated with 30 µM LPA for 12 h (n = 3). B Western blot images and quantification of PKC?, P-ERK1/2 and P-c-Jun protein expression in KCs treated with LPA (30 µM 12 h) (n = 3). C Western blot images and quantification of GPAT3, PKC?, P-ERK1/2 and P-c-Jun protein expression in LPS-stimulated KCs transfected with GPAT3 siRNA (n = 3). D Western blot images and quantification of inflammatory cytokines and ERK1/2 pathway expression in control or LPA treatment KCs and pretreatment U0126 (10 µM 2 h, ERK inhibitor) (n = 3). Data represents mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 4: Exogenous LPA enhanced inflammatory response in vitro and vivo.A Effect of si-GPAT3 or si-N.C. on TG content in KCs stimulated with LPS (1 µg/ml) for 24 h (n = 3). B Bodipy 493/503 fluorescence visualized by fluorescence microscopy (scale bars represent 50 µm) (n = 3). C LPA concentrations in the supernatant of KCs after LPS (100 ng/ml 12 h) stimulated and with or without si-GPAT3 (n = 3). D The mRNA expression of IL-1ß, NLRP3, IL-1a and IL-6 in KCs treated with LPA (30 µM 12 h) (n = 3). E, F The effects of exogenous LPA (30 µM 12 h) on the protein expression of inflammatory factors IL-1ß, NLRP3, COX2, IL-1a and IL-6 in KCs (n = 3). G Experimental design of mice treated with LPA. H, I The contents of LDH, IL-6 and IL-1ßin plasma of LPA treated with mice (n = 6). J, K Representative images and quantification of HE staining (Scale bars represent 100 µm) and CD68 IHC (Scale bars represent 200 µm) after LPA treatment in liver of mice (n = 3). Data represents mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 5: Blocking GPAT3 decreases inflammatory response in LPS-stimulated Kupffer cells.A The mRNA expression of IL-1a, IL-6, IL-1ß and NLRP3 in si-N.C. or si-GPAT3 KCs with or without LPS (100 ng/ml, 12 h) (n = 3). B, C The protein levels of IL-1ß, NLRP3, COX2, IL-1a and IL-6 in si-N.C. or si-GPAT3 KCs with or without LPS (100 ng/ml, 12 h) (n = 3). D Heatmap showing the inflammatory cytokines genes expression in LPS-stimulated KCs with or without si-GPAT3 as measured by transcriptomics (n = 3). E Scatter plot of GO enrichment differential genes of si-N.C. vs si-GPAT3 (n = 3). F The expression of GPAT3 in KCs treated with FSG67 (150 µM, 1 h) was analyzed by Western blot and qPCR (n = 3). G, H The levels of IL-1ß and IL-6 in KCs were pretreated with 150 µM FSG67 for 6 h and then were treated with 100 ng/mL LPS for 6 h (n = 3). Data represents mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.