Journal: International Journal of Biological Sciences

Article Title: TM4SF5-mediated KEAP1 Regulation in Hepatocytes Irrelevant to NRF2 Expression and Activity Promotes Oxidative Stress and Inflammation to Develop Metabolic Dysfunction-Associated Steatotic Liver Disease

doi: 10.7150/ijbs.126251

Figure Lengend Snippet: TM4SF5-mediated stabilization of KEAP1 in lipid-treated hepatocytes promotes oxidative stress and hepatic inflammation. (A-F) Subconfluent Huh7 Control or Huh7 TM4SF5-KO hepatocyte variants, either stably or transiently transfected with the indicated cDNAs, were analyzed for ROS production using flow cytometry following DCFDA staining. Cells received vehicle treatment (-), PA alone (B, D, and F), lipid mixture (LM, C), or DOX to induce Keap1 knockdown (shKEAP1 #2 or #3 , see Table , C and D). In some experiments, Huh7 cell variants were transfected with siNS (non-specific sequences as a control) or siNRF2 (targeting sequence #1 or #2, see Table ) for 24 h. Cells were then treated with DOX for shKEAP1 #2 induction for 24 h, followed by PA for 4 h prior to ROS analysis by flow cytometry (D). Cells were also exposed to H 2 O 2 at the indicated concentrations and durations before assessment of ROS levels (E). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ***, P < 0.0001; ns = non-significant, unpaired Student's t test or two-way ANOVA. Data are expressed as mean ± SEM. (G) Subconfluent SNU449 hepatocytes stably expressing empty vector (SNU449 EV ) or TM4SF5 (SNU449 TM4SF5 ) were harvested to perform qRT-PCR for the indicated molecules. (H) Subconfluent Huh7 Control cells were treated in the absence (-) or presence (+) of DOX to induce KEAP1 knockdown (shKEAP1 #2 ) for 24 h and then exposed to PA (100 μM) for 4 h before being collected for immunoblot analysis of the indicated molecules. (I) Murine AML12 cells stably transfected with EV or TM4SF5 were treated with 5% LM for 24 h, harvested, and subjected to qRT-PCR for the indicated molecules. Data shown are representative of three independent experiments. See also .

Article Snippet: The murine normal hepatocyte AML12 cell line, lacking TM4SF5, was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Control, Stable Transfection, Transfection, Flow Cytometry, Staining, Knockdown, Sequencing, Expressing, Plasmid Preparation, Quantitative RT-PCR, Western Blot