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. 2020 Mar 9;11(3):178. doi: 10.1038/s41419-020-2369-4

Fig. 6. TNFAIP8 binds with oleic acid and enhances HCC cell steatosis.

Fig. 6

a The location of the coiled-coil domain, D-Box consensus, and the positions of cis-/trans-OA binding key amino acids of TNFAIP8 are shown. b Molecular modeling images of cis- and trans-oleic acid binding with mouse TNFAIP8 are shown (left and right panels). c The purity of TNFAIP8-His-tagged protein and specificity of TNFAIP8 antibody was examined by simplyblue staining and immunoblotting. d Binding of TNFAIP8 with fatty acids was determined by ELISA as described in materials and methods section. OA: oleic acid, EA: elaidic acid, PA: palmitic acid, LA: lauric acid. e HCC cells were grown on coverslips and transfected with EV or TNFAIP8-Myc plasmid and treated with 100 µM oleic acid for 24 h. Cells were stained with Oil Red O (ORO) and anti-Myc rabbit antibody overnight, followed by Alexa-Fluor-568-conjugated anti-rabbit secondary antibody. After washing, cells were stained with DAPI, mounted, and photographed (arrow indicate lipid droplets in TNFAIP8 overexpressing cells). f EV and TNFAIP8-Myc-stable-expressing HepG2 cells were grown on coverslips and treated with 100 µM oleic acid for 24 h and stained with ORO (upper panel). The number of ORO-stained lipid droplets present in the EV and TNFAIP8-stable-expressing cells was measured and plotted (lower panel). g HCC cells were grown in 6-well plates and transfected with EV or TNFAIP8-Myc plasmids for 30 h and treated with 100 µM oleic acid for 24 h and lysates were WB with indicated antibodies. h Similarly, transfected and treated cells were stained with ORO, cells were lysed, and ORO stain released from steatotic cells was measured and plotted. i HCC cells were transfected with control siRNA or TNFAIP8 siRNA for 30 h and lysates were WB with indicated antibodies. j Control siRNA or TNFAIP8-siRNA transfected cells were treated with 100 µM oleic acid for 24 h, and after Oil Red O staining, cells were lysed, and ORO stain released from steatotic cells was measured and plotted. All data represent mean ± SEM from two (d, j, h) independent experiments in triplicate. $$$P < 0.001 relative to EV transfected. **P < 0.01, ***P < 0.001 relative to EV transfected and OA treated cells. #P < 0.05, ###P < 0.001 relative to control siRNA transfected cells. EV: empty vector, OA: oleic acid.