Fig. 5. DAP3185S is a key site that modulates mitochondrial function and HCC progression.
A Huh7 cells were transfected with the DAP3 overexpression vector (OE-DAP3), DAP3S185A overexpression vector (OE-S185A), DAP3S31A overexpression vector (OE-S31A) or control vector (Con) for 48 hours. MT-ND5 protein expression was measured using western blotting. B–L Huh7/Hep3B cells with stable DAP3 knockdown (KD) and the corresponding negative control (NC) Huh7/Hep3B cells were transfected with the DAP3 overexpression vector (OE), DAP3S185A overexpression vector (Mut-OE) or control vector (Con) for 48 h. B MT-ND5 protein expression was measured using western blotting. C Mitochondrial ETC complex I enzyme activity levels in the indicated groups (representative of three independent experiments). D Representative OCR measurements in the indicated groups upon the addition of oligomycin, the mitochondrial decoupler FCCP, and rotenone + antimycin. Basal and maximal respiration and ATP production were quantitatively calculated. E The NAD+/NADH ratio was determined by performing a colorimetric WST-8 assay (representative of three independent experiments). F ATP production was determined using an ATP Bioluminescent Assay Kit (representative of three independent experiments). G Flow cytometry analysis of the mitochondrial mass and mitochondrial membrane potential after labelling cells with MitoTracker Green and MitoTracker DeepRed (representative of three independent experiments). H CCK-8 assays were used to evaluate the proliferative capacity of Huh7 cells after the indicated treatments (representative of three independent experiments). I EdU incorporation assays (EdU, red; Hochest, blue) were performed to analyse the cell cycle after the indicated treatments and compare the percentage of S phase cells. Scale bars, 50 μm. Hoechst staining was used to indicate total cells, and EdU incorporation indicated cells with active DNA replication. J The relative expression of proliferation-related genes and proteins (Ki67 and PCNA) was verified using qPCR and western blotting (representative of three independent experiments). K Transwell assays were used to measure the migration and invasion of Huh7 cells after the indicated treatments. Scale bars, 200 μm. The bar charts show the relative counts (relative to the NC group) of cells that passed through the chamber membrane in each group (right panel) (representative of three independent experiments). L Western blot analysis of EMT-related proteins in Huh7 and Hep3B cells subjected to the indicated treatments (representative of three independent experiments). M The phosphorylation of wild-type or mutant DAP3 in the cytoplasm and mitochondria of Huh7 cells was detected using IP. GAPDH was used as a loading control for cytoplasmic proteins, and TOMM20 was used as a loading control for mitochondrial proteins. N Western blot analysis of DAP3 expression in the cytoplasm and mitochondria of cells treated with an AKT inhibitor (10 μmol/L for 24 h). O Western blot analysis of MT-ND5 expression in Huh7 cells transfected with OE-DAP3 or OE-S185A or treated with an AKT inhibitor (10 μmol/L for 24 hours). Statistical analysis was performed using two-way ANOVA (Fig. 5H) or 2-tailed Student’s t test (Fig. 5C, D, E, F, G, I, J, and K). The error bars indicate the means ± SDs. n.s., not significant; *p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001.