Fig. 6: HMGB1 is required to induce hepatocyte senescence during IRI.

A Schematic representation showing hiPSC-Heps hepatocytes treated with soluble HMGB1, TNFα, or a combination of both during reperfusion. B Intracellular ATP quantification in hiPSC-Heps treated with soluble HMGB1, TNFα or a combination of both during reperfusion (n = 4). Data are represented as % of ATP in the experimental conditions vs. control (not induced to IRI). C Schematic representation of the co-cultured M1- primary macrophages with hiPSC-Heps and subjected to IRI with neutralizing antibody anti-HMGB1 or anti-TNFα during reperfusion. D Intracellular ATP quantification in hiPSC-Heps treated with neutralizing antibody anti-HMGB1 or anti-TNFα during reperfusion (n = 4). E Representative pictures of hiPSC-Heps co-cultured with activated M1- primary macrophages for 24 h of reperfusion and stained for β-Galactosidase (Scale bar: 100 μm). F Senescence quantification of β-Galactosidase+ hiPSC-Heps co-cultured with activated macrophages at 24 h of reperfusion. G Representative immunoblot of p16 and p21 in hiPSC-Heps treated with neutralizing antibody anti-HMGB1 or anti-TNFα and subjected to IRI. p16 and p21 protein levels were analyzed at 24 h reperfusion. Calnexin protein (CANX) was used as loading control. H p16 and p21 protein quantification from hiPSC-Heps treated with neutralizing antibody anti-HMGB1 or anti-TNFα and subjected to IRI. p16 and p21 proteins were analyzed at 24 h reperfusion. D, H, I One-way ANOVA with Tukey correction, F unpaired t-tests (*p < 0.05; ****p < 0.005; ns not significant). Created in BioRender. Zito, G. (2025) https://BioRender.com/ahmrf54.