Fig 1: TNFAIP8-autophagy axis is associated with hepatic steatosis induced by EtOH.a HCC cells were treated with 50 mM EtOH for 30 h, and lysates were immunoblotted with indicated antibodies. b HCC cells were transfected with EV or TNFAIP8-Myc plasmid for 18 h and treated with 50 mM EtOH for 24 h and lysates were immunoblotted indicated antibodies. c HCC cells were grown on a coverslip and transfected with EV or TNFAIP8-Myc plasmid for 18 h and treated with 50 mM EtOH and 100 µM OA for 24 h. Cells were stained with Oil Red O and TNFAIP8-Myc protein as described in materials and methods section and observed under an Olympus BX60 fluorescent microscope with the bright field (×40 objective) and photographed. Arrow indicates TNFAIP8 expression and cell steatosis. d Representative hematoxylin and eosin (H&E, original magnification ×400) stained liver sections of male C57BL/6J mice pair-fed chronic ethanol (EtOH) or control diets for 8 weeks. e C57BL/6J mice pair-fed chronic ethanol (EtOH) or control diets for 8 weeks, mice were euthanized, and 50 µg of liver lysates were immunoblotted with indicated antibodies (upper panel). * = non-specific band. Indicated protein levels from the upper panel were quantified using ImageJ software (https://imagej.nih.gov/ij/) and plotted (lower panel). $P < 0.05 relative to control mice. f TNFAIP8 expression is higher in hepatic steatosis human patients with a history of alcohol use: left and right panels: representative images and quantification of TNFAIP8 expression in liver tissue, including normal liver tissues, liver steatosis without alcohol use, and liver steatosis with a history of alcohol use. g Left panel: Representative images of TNFAIP8 expression in normal liver, steatohepatitis (NASH) with no history of alcohol use, and steatohepatitis with alcohol use. Right panel: the overall quantification of TNFAIP8 expression in normal liver tissue, liver steatosis without a history of alcohol use, and liver steatosis with a history of alcohol use. TNFAIP8 expression was analyzed and quantified from TMA. Boxplots show median and interquartile range with whiskers indicating the total range of TNFAIP8 expression. Data are expressed as the mean ± SEM from 6 (e) mice or mean from 5–18 (f, g) human samples. *P < 0.05 relative to normal liver. ns: not significant.
Fig 2: TNFAIP8 inactivates AKT/mTOR and induces autophagy.a Cell lysates from HepG2 and SK-Hep1 cells transfected with EV and TNFAIP8-Myc plasmids (left and middle panels) and EV or TNFAIP8-Myc-stable-expressing HepG2 cells (right panel) were WB with indicated antibodies. Indicated protein levels were quantified using ImageJ software (https://imagej.nih.gov/ij/). b HCC cells were treated with TNFa (10 to 50 ng/ml) for 30 h and lysates were WB with indicated antibodies. c, d HepG2 and SK-Hep1 cells were pretreated with 3-MA (2 mM) or chloroquine (10 µM) for 8 h and cells were transfected with EV or TNFAIP8-Myc plasmid in the presence of the indicated autophagy inhibitor for 40 h, and cell survival was measured by MTT assay. e, f HepG2 and SK-Hep1 cells were pretreated with 3-MA (2 mM) for 8 h, the cells were transfected with EV or TNFAIP8-Myc plasmid in the presence of autophagy inhibitor for 24 h and then treated with sorafenib (5 µM) or regorafenib (0.5 µM) for additional 40 h, and cell survival was measured by MTT assay. Data represent mean ± SEM from four (d–f) independent experiments. ***P < 0.001, ###P < 0.001 compared with EV transfected. aaaP < 0.001, ßßßP < 0.001 compared with TNFAIP8 transfected. EV: empty vector, NS: not significant, 3-MA: 3-methyladenine, Chlor: chloroquine, Sora: sorafenib, Rego: regorafenib.
Fig 3: TNFAIP8 binds with oleic acid and enhances HCC cell steatosis.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.
Fig 4: TNFAIP8 interacts with ATG7-ATG3 complex.a HepG2 cell lysates (1 mg) were incubated with pure TNFAIP8 protein (2 µg) for 3 h. TNFAIP8 was pulled-down with TNFAIP8 antibody or control IgG. Immunocomplexes were resolved by SDS-PAGE and WB with indicated antibodies. b, c Interaction of TNFAIP8-Myc protein with ATG7-ATG3-LC3B complex was analyzed by forward and reverse IP as indicated. d Effect of TNFa on cellular ROS production was visualized by staining the cells with CellROX Green reagent (Invitrogen). e Effect of TNFa on TNFAIP8 and IL-6 mRNA expression was analyzed by RT/qPCR (n = 3). f, g Effect of TNFa on ATG3, ATG7. TNFAIP8 and LC3B protein expression were analyzed by WB and interaction of TNFAIP8 with ATG3-ATG7 was analyzed by TNFAIP8 IP followed by WB. *P < 0.05, **P < 0.01 relative to untreated SK-Hep1 cells. IP: immunoprecipitation, WB: western blotting.
Fig 5: TNFAIP8 induces liver cancer cell survival and drug resistance by reducing apoptosis.a WB analysis of TNFAIP8-Myc tagged protein expression in HCC cells. b Effect of overexpression of TNFAIP8 on HCC cell survival was measured by MTT assay. c Effect of overexpression of TNFAIP8 on liver cancer cell colony formation. d Effects of sorafenib or regorafenib on HepG2 cell survival transfected with EV or TNFAIP8-Myc-tagged plasmids were measured by MTT assay. e Stable expression of TNFAIP8 in HepG2 was analyzed by WB. f EV and TNFAIP8-Myc-stable-expressing cells were treated with sorafenib (5 µM) or regorafenib (0.5 µM) for 48 h, and expression of TNFAIP8-Myc tagged protein was analyzed by WB. g, h Effects of sorafenib (5 µM) or regorafenib (0.5 µM) on EV and TNFAIP8-Myc-stable-expressing HepG2 cells on survival and cell colony formation were measured. i WB analysis of the effect of sorafenib, regorafenib, and doxorubicin on cell apoptosis markers expression in HepG2 cells transfected with EV or TNFAIP8-Myc-tagged plasmids. j HepG2 and SK-Hep1 cells were transfected with control siRNA or TNFAIP8 siRNA for 30 h, and cell lysates were WB with anti-TNFAIP8 and anti-GAPDH antibodies. k Control siRNA and TNFAIP8-siRNA transfected cells (10,000) were re-plated in 96-well plates and treated with sorafenib (5 µM) or regorafenib (0.5 µM) for 48 h, and cell survival was measured by MTT assay. Data represent mean ± SEM from two to three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 relative to EV transfected, and Sora or Rego-treated. ???P < 0.001 compared with EV stably transfected. ###P < 0.001 relative to EV stably transfected and Sora treated. ???P < 0.001 compared with EV transfected and Rego-treated. aaaP < 0.001, ßßßP < 0.001 compared with control siRNA transfected. $$$P < 0.001 compared with control siRNA transfected and Sora treated. †††P < 0.001 relative to control siRNA transfected and Rego-treated. EV: empty vector, Sora: sorafenib, Rego: regorafenib, NS: not significant, WB: western blotting.
Supplier Page from Sino Biological, Inc. for Human TNFAIP8 Protein (His Tag)