Fig. 1: Intracellular distribution pattern of NOTCH1 intracellular domain (NICD1) in mouse cardiac endothelial cells and cultured cell lines.

A Representative western blot illustrating NICD1 levels in subcellular fractions from rat primary cardiac endothelial cells. Mitochondrial (Cytochrome C oxidase subunit 4, COX 4), nuclear (Histone H3, H3), and cytoplasmic (glyceraldehyde-3-phosphate dehydrogenase, GAPDH) markers were used. Three experiments were repeated independently with similar results. B Top: representative western blot illustrating NICD1 levels in whole-cell lysates and subcellular fractions from mouse primary cardiac endothelial cells and cardiomyocytes from mouse embryo hearts at E11.5 and E14.5. Bottom: Relative levels of mitochondrial NICD1 in mouse primary cardiac endothelial cells and cardiomyocytes (n = 3). Data are presented as mean values ± SD. P values were calculated by unpaired Student’s t test with two-tailed analysis without adjustments. C Representative western blot illustrating NICD1 levels in cultured human umbilical vein endothelial cell (HUVEC), human embryonic kidney 293 cells (HEK293T), MKN45 gastric cancer cells, and BxPC-3 pancreatic cancer cells. Three experiments were repeated independently with similar results. D, E Immunofluorescence confocal microscopy images showing the colocalization of NOTCH1 (green) and mitochondria (red) in HUVEC and HEK293T cells. Results are representative of three different experiments. Scale bars: 20 μm for HUVEC and 10 μm for HEK293T. F Location and sequences of putative internal signals (M1–8) in NICD1. RAM Rbp-associated molecule domain, ANKs ankyrin repeats, TAD transcription activation domain, PEST proline (P), glutamic acid (E), serine (S), threonine (T) degradation domain, NLS nuclear localization signals. G Left: representative western blot of NICD1 levels in subcellular fractions from HUVEC and HEK293T cells overexpressing FLAG-tagged wild-type NICD1 or internal signal-deleted NICD1 (ΔM1–8). Three experiments were repeated independently with similar results. Right: Ratio of NICD1 level in mitochondria to whole-cell lysate in HUVEC and HEK293T cells. Source data are provided as a Source Data file.