Fig. 5: Hepatocyte TIA1 is essential for SGs formation during PA-induced hepatic injury.

A Representative immunofluorescence images of AML12 cells following genetic manipulation. Cells were infected with adenovirus overexpressing TIA1 (Ad-TIA1) or GFP control (Ad-GFP), or transfected with shRNA targeting TIA1 (shTIA1) or negative control shRNA (shNC), then treated with PA (0.3 mM for 24 hours). Cells were co-stained for TIA1 (green) and the core SGs marker G3BP1 (red). Nuclei were counterstained with DAPI (blue). White boxes in indicate regions shown representative SGs exhibiting co-localization. Scale bar: 10 μm. B Quantification of SGs formation from experiments in (A). Left panel: Percentage of cells containing SGs (n = 50 cells per group). Right panel: Average number of TIA1 G3BP1 double positive SGs per cell (n = 50 views per group). C–E AML12 cells were infected with shTIA1 or shNC, with or without pre-treatment with the SGs inducer sodium arsenite (Ars, 0.5 mM, for 1 hour), followed by PA challenge (0.3 mM for 24 h). C Representative ORO staining visualizing intracellular lipid droplets in indicated groups and the normalized quantification (n = 5 independent biological replicates; mean ± SEM). Scale bar: 50 μm. D Western blot analysis of lipogenic proteins (FASN, SREBP1, SCD1, PPARγ). GAPDH served as a loading control. E qRT-PCR analysis of lipid metabolism-related gene expression (Fasn, Srebf1, Scd1 and Pparg), normalized to Gapdh (n = 5 independent biological replicates; mean ± SEM). F–H AML12 cells were infected with Ad-TIA1 or Ad-GFP, with or without pre-treatment with the SGs inducer anisomycin (An, 25 ng/mL, for 30 min), followed by PA challenge (0.3 mM for 24 h). F Representative ORO staining and normalized quantification (n = 5 independent biological replicates; mean ± SEM). Scale bar: 50 μm. G Western blot analysis of lipogenic proteins (FASN, SREBP1, SCD1, PPARγ). GAPDH served as a loading control. H qRT-PCR analysis of lipid metabolism-related gene expression (Fasn, Srebf1, Scd1 and Pparg), normalized to Gapdh (n = 5 independent biological replicates; mean ± SEM). *P < 0.05, **P < 0.01, ***P < 0.001; ****P < 0.0001; ns indicates not significant.