Fig. 2

G6PD translocates to mitochondria and affects mitochondrial function. A Mitochondrial and cytoplasmic fractions were prepared from VSMCs treated with 20 ng/mL PDGF-BB for 12 h. Western blot analysis of the cytosolic and mitochondrial fractions was performed to evaluate the translocation of G6PD from the cytosolic compartment to the mitochondria. GAPDH and TOM40 were used as the cytosolic and mitochondrial loading controls, respectively. B Immunofluorescence analysis was carried out after 12 h of PDGF-BB stimulation, and 6AN and siG6PD were also used in subsequent experiments. Mitochondria were identified with TOM40, nuclei were stained with DAPI, and a G6PD monoclonal antibody was used to indicate endogenous G6PD. Scale bar = 50 µm. n = 3. C, D Seahorse metabolic flux analyses showing the traces and quantification of the basal or maximum mitochondrial oxygen consumption rate (OCR, C) and extracellular acidification rate (ECAR, D) in VSMCs treated with 6AN and siG6PD under PDGF-BB stimulation. n = 3. Statistical significance was determined using two-tailed Student’s t tests in (A) and one-way ANOVA in (B–D). *P < 0.05; **P < 0.01; ***P < 0.001