Fig. 4: The small-molecule pyruvate dehydrogenase kinase isoform 4 (PDK4) inhibitor GM10395 promotes metabolic shift toward oxidative phosphorylation in inflammatory cytokine mixture (ICM)-treated human retinal pigment epithelium (hRPE) cells.

A, B Protein was isolated from ICM-treated primary hRPE cells ± dichloroacetate (DCA; 1 and 10 μM) or GM10395 (0.5 and 1 μM) for 24 h. Lysates were subjected to immunoblotting for phosphorylated pyruvate dehydrogenase E1-alpha subunit (p-PDHE1α), and heat shock protein 90 (HSP90) ratios were determined by densitometry (n = 3 replicates/experiment). C Oxygen consumption rate (OCR) was measured in primary hRPE cells with ICM treatment for 24 h on day 3 ± DCA (1 and 10 μM) or GM10395 (0.5 and 1 μM). Basal respiration, maximal respiration, spare respiratory capacity, and ATP production rate were calculated based on the OCR response to specific inhibitors (n = 5/group). D Mitochondrial superoxide (MitoSOX) levels were measured in primary hRPE cells with ICM treatment for 24 h ± DCA (10 μM) or GM10395 (1 μM). Scale bar: 50 μm. Quantification of MitoSOX intensity (n = 27 microscopic fields with > 4 × 10⁴ cells counted from three independent wells). E Primary hRPE cells treated with ICM ± DCA (10 μM) or GM10395 (1 μM) for 24 h were fixed and stained with translocase of outer membrane 20 (TOM20) antibody to visualize mitochondrial membranes. Scale bar: 20 μm. Inset scale bar: 5 μm. Quantification of mitochondrial area, fragmentation, and elongation of > 65 cells counted for each condition. Data are represented as mean ± SEM. ^#P < 0.05; ^##P < 0.01; ^###P < 0.001 versus control; *P < 0.05; **P < 0.01; ***P < 0.001 versus ICM group. One-way ANOVA with Tukey’s multiple comparisons test.