Fig. 4: MASLD phenotypic spectrum in Condition 2 hepatocytes.

A Male (donor SMC) and female (donor HU8406) intracellular triglycerides, normalized to total protein abundance, are significantly elevated in Condition 2 hepatocytes compared with Condition 1. B Abundance of primary conjugated bile acids is significantly increased at day 9 in Condition 2 vs. Condition 1. After 18 days in culture, taurocholic acid is still significantly elevated in Condition 2 over the Condition 1 stimulated male hepatocytes. C CCL2, IL8, CXCL1, and CXCL10 secretion at days 3, 9, 14, and 19 was assessed in Condition 1 and Condition 2 male and D female hepatocytes within our MPS model, revealing a pro-inflammatory state in hepatocytes stimulated with physiologically high amounts of insulin, glucose, and FFA. E Multiphoton label-free imaging of male Condition 1 and Condition 2 hepatocytes using third harmonic generation (THG; Lipids), three-photon autofluorescence (3PAF; NAD(P)H), and two-photon autofluorescence (2PAF; FAD). F Quantitation of label-free imaging revealed increased intracellular lipid content in Condition 2 vs Condition 1 hepatocytes. * p < 0.05, ** p < 0.01 *** p < 0.001, **** p < 0.0001; Student’s t-test or two-way ANOVA. Scale bar: 50 µm.