Fig. 3: Mitochondria-located NOTCH1 intracellular domain (NICD1) activation of pyruvate dehydrogenase (PDH) and mitochondrial respiration.

A Enzymatic activity of PDH, CS, and IDH in mouse cardiac ECs isolated from mice (n = 5). B Enzymatic activity of PDH, CS, and IDH in cells (n = 5). C Enzymatic activity of PDH, CS, and IDH in cells transfected with siNC or NOTCH1 siRNA (siNOTCH1) (n = 5). D Top: schematic of recombinant mitoNICD1 protein containing a presequence of cytochrome oxidase subunit 8 (COX8). Bottom: representative western blot illustrating intracellular distribution of wild-type NICD1 and mitoNICD1 (left) and quantification of mitochondrial NICD1 (n = 3) (right). E Representative immunofluorescence confocal microscopy illustrating intracellular distribution of NICD1 and mitoNICD1 (left) and the quantification of mitochondrial NICD1 (n = 3) (right) (Scale bar = 20 μm). F Representative western blot illustrating NICD1 levels (Top) and PDH activities (Bottom) in cells with different treatments (n = 5). G Relative ratio of citrate to pyruvate in wild-type (WT) cells, NOTCH1 KO cells and NOTCH1 KO cells expressing mitoNICD1 (n = 4). H Relative lactate level in WT cells, NOTCH1 KO cells and NOTCH1 KO cells expressing mitoNICD1 (n = 4). I Relative ratio of citrate to pyruvate in mouse cardiac endothelial cells isolated from wild-type and Notch1^+/− mice (n = 10). J Schematic of possible fates for [U-¹³C]glucose. K Fraction enrichment of lactate [m+3], alanine [m+3], acetyl-CoA [m+2], and citrate [m+2] in cells expressing exogenous wild-type NICD1 or not (n = 5). L Fraction enrichment of lactate [m+3], alanine [m+3], acetyl-CoA [m+2], and citrate [m+2] in cells transfected with scramble siNC or siNOTCH1 (n = 5). M Left: profile of oxygen consumption rate (OCR) of WT cells, NOTCH1 KO cells, and NOTCH1 KO cells expressing mitoNICD1. Oligomycin (Oligo), 2-[2-[4-(trifluoromethoxy)phenyl] hydrazinylidene]-propanedinitrile (FCCP), antimycin and rotenone (Rot/AA) were supplemented at 20, 50, and 80 min during the test. Right: Quantification of basal respiration (n = 9) and maximal respiration indicated by OCR (n = 6). N ATP levels of WT cells, NOTCH1 KO cells, and NOTCH1 KO cells expressing mitoNICD1 as indicated by luminescence (n = 6). Source data are provided as a Source Data file. For A–I and K–N, data are presented as mean values ± SD; P values were calculated by unpaired Student’s t-test with two-tailed analysis without adjustments.