Extended Data Fig. 3: Chemogenetic mitochondrial H2O2 regulates endothelial NO signaling through IL-6-HDAC4 pathway.
a, Schematic diagram of the experimental setting: PECs were transduced with lentiviral vectors encoding D-amino acid oxidase (DAAO)-HyPer7.2 targeted to the cell mitochondria for generation of mitochondrial H2O2 in the presence of D-alanine (10 mM) for 72 h. b, Representative widefield ratiometric HyPer7.2 images of PECs transduced with Lenti-DAAO-HyPer7.2-mito constructs. c-i, Immunoblots (c) and quantitative plots show that chemogenetic mitochondrial H2O2, produced by DAAO in response to D-alanine but not L-alanine, orchestrates IL-6-mediated CaMKII-HDAC4 phosphorylation and the subsequent VCAM1-regulated eNOS phosphorylation at Ser1177 and Thr495 in PECs (n = 4–6). Scale bar, 100 μm. Error bars represent SD (d–i). Data represent triplicated biologically independent experiments. P values were calculated using a two-tailed unpaired Student’s t-test (d–i). Images created with BioRender.com (a). (*P < 0.05, **P < 0.01, ***P < 0.001).
