Fig. 3

MCU deficiency attenuates mitochondrial Ca²⁺ uptake, oxidative stress, and mitochondrial dysfunction in AML12 hepatocytes under lipotoxicity. A Representative images of mitochondrial Ca²⁺ fluorescence (Rhod-2 Ca²⁺ indicators) in AML12 hepatocytes. Scale bars: 5 μm. B Rhod-2 Ca²⁺ quantified using ImageJ software (n = 9 fields). C ROS levels assessed by flow cytometry using H₂DCFDA in AML12 hepatocytes and signal was measured by flow cytometry. D Representative image of MitoSOX in AML12 hepatocytes. Scale bars: 20 μm. E Mitochondrial membrane potential (MMP, Δψm) in AML12 hepatocytes was detected by JC-1 dye staining and visualized with a fluorescence microscope (×400). Scale bars: 50 μm. F Quantification of mitochondrial ROS (miROS) based on MitoSOX Red fluorescence (D). The data were pooled from three independent experiments. G Quantification of JC-1 fluorescence in AML12 hepatocytes. The data were pooled from three independent experiments. H Relative intracellular ATP content. I qRT-PCR analysis of Pgc1α mRNA expression. Results were normalized to β-actin mRNA and expressed as fold change compared to control group. Data are mean ± SD. Two-way ANOVA with Tukey’s test (B, C, F, G, H) and two-way ANOVA with Sidak’s test (I). **P < 0.01, ***P < 0.001, and ****P < 0.0001; ns indicates not significant