Fig. 5: Mitochondria-targeted NOTCH1 intracellular domain (mitoNICD1)-induced pyruvate dehydrogenase (PDH) activation promotes endothelial-to-mesenchymal transition.

A Left: representative immunofluorescence confocal microscopy showing the endothelial cell marker CD31 (red) and the mesenchymal cell marker α smooth muscle actin (α-SMA) (green) in human umbilical vein endothelial cells (HUVEC) treated with cytokines (10 mg/μL transforming growth factor β [TGF-β1] and 1 ng/μL interleukin-1β [IL-1β]), expressing exogenous NOTCH1 intracellular domain (NICD1), or mitoNICD1 in low-power field (LPF) (10×) (top, scale bar=200 μm) and high-power field (HPF) (×40) (bottom, scale bar = 50 μm)). Nuclei are stained with DAPI (blue). Right: Quantification of α-SMA positive (α-SMA⁺) cells per LPF (n = 5 biological samples per group). B Representative western blot illustrating endothelial cell markers, including CD31, vascular endothelial cadherin (VE-Cad), and vascular endothelial growth factor receptor (VEGFR), and mesenchymal cell markers, including α-SMA, N-Cadherin (N-Cad), Vimentin, and Actin, in HUVEC expressing exogenous NICD1, mitoNICD1, or not. C Representative western blot illustrating the phosphorylation of pyruvate dehydrogenase E1 subunit alpha 1 (PDHA1) (p-PDHA1) at serine 293 (Ser293) in HUVEC treated with PDH-activator dichloroacetate (DCA) (10 mmol/L), expressing mitoNICD1, or expressing mitoNICD1 and supplemented with PDH inhibitor PS-48 (100 μmol/L) as well. D Top: profile of oxygen consumption rate (OCR) of HUVEC treated with DCA (10 mmol/L), expressing mitoNICD1, or expressing mitoNICD1 and supplemented with PS-48 (100 μmol/L) as well. Bottom: Quantification of basal respiration and maximal respiration indicated by OCR (n = 3 biological samples per group). E Left: representative immunofluorescence confocal microscopy showing CD31 (red) and α-SMA (green) in HUVEC treated with DCA (10 mmol/L), expressing mitoNICD1, or expressing mitoNICD1 and supplemented with PS-48 (100 μmol/L) as well (Scale bar=200 μm). Right: quantification of α-SMA⁺ cells per LPF (n = 5 biological samples per group). F Representative immunofluorescence confocal microscopy of CD31 (red) and α-SMA (green) in HUVEC treated with 5, 10, or 20 mmol/L DCA (Scale bar = 200 μm). Right: quantification of α-SMA⁺ cells per LPF (n = 5 biological samples per group). Source data are provided as a Source Data file. For A, D–F, data are presented as mean values ± SD; P values were calculated by unpaired Student’s t test with two-tailed analysis without adjustments.