Fig. 5: Inhibition of mTORC2 promoted ROS accumulation and eNOS uncoupling.

a HAEC were treated with 1 nM rapa or 10 nM torin for 1 h followed by incubation with dihydroethidium (DHE) for detection of intracellular superoxide with flow cytometry (n = 5). b HAEC were transfected with siRNA prior to flow cytometric measurement of intracellular superoxide (n = 5–9). c Primary endothelial cells (EC) were isolated from Mtor^EC−/− or Rictor^EC−/− mice and their WT littermates to measure intracellular superoxide (n = 5–6). d Serum superoxide levels were measured in Mtor^EC−/−, Rpt^EC−/− and Ric^EC−/− mice with chemiluminescent assay (n = 7–9). e HAEC were treated with 1 nM rapa or 10 nM torin for 1 h followed by ELISA measurement of BH4 (n = 4–5). f After treatment, eNOS dimer (di) to monomer (mo) ratio was evaluated by native PAGE followed by western blotting (n = 4). g After pretreatment with L-NAME (500 nM) for 0.5 h (n = 3–6), HAEC were incubated with 10 nM torin for 1 h prior to measurement of intracellular superoxide with flow cytometry. h, i After 1 h pretreatment with N-acetylcysteine (Nac, 2 mM), MitoQ (1 µM), Apocynin (Apo, 20 µM), or Allopurinol (All, 50 µM) (n = 3–4), HAEC were incubated with 10 nM torin for 1 h prior to measurement of superoxide inside HAEC (f) and NO in the medium (g). j HAEC were treated with 10 nM torin for 1 h prior to quantitative PCR (n = 5). k, l HAEC were treated with 10 nM torin or transfected with siRNA followed by Western blotting to quantify Nox2 expression (n = 4). Error bars correspond to standard error of the mean (SEM). *p < 0.05; **p < 0.01 vs. ctrl, WT, sictrl or torin-treated ctrl; one-sample t test (a, c, d, g, j, k) or RM one-way ANOVA with Dunnett’s test (b, f, h, l) or one-way ANOVA with Dunnett’s test (e, i).