Figure 3: Characterization of WT, Nox2-deficient (Nox2^y/−) and TRPC6-deficient (TRPC6^−/−) primary MLEC.

(a) Identification of MLEC (WT, wild-type; TRPC6^−/−, TRPC6-deficient; Nox2^−/−, Nox2-deficient; 2nd ab only, only second antibody applied) cultured for 7 days. Cells were stained with antibodies against platelet endothelial cell adhesion molecule-1 (PECAM-1 ab). Scale bars correspond to 200 μm. (b) Immunoblotting of cell lysates extracted from WT and TRPC6^−/− MLEC, and detection of TRPC6 protein using a TRPC6-specific antiserum. (Lower part) An imunoblot detecting vinculin serves as a loading control. (c) Extracellular superoxide release after 30 min of ischaemia or normoxia in MLEC. (d) Differences in intracellular H₂O₂ levels in MLEC induced by ischaemia–reperfusion and detected by the HyPer sensor. (e) Increase in the [Ca²⁺]_i upon ischaemia–reperfusion (Norm., Perf.). (f) Cation influx in MLEC from WT and TRPC6^−/− mice. Increase in the [Ca²⁺]_i upon ischaemia–reperfusion (Norm., Perf.). (g) Ischaemia-induced Mn²⁺ influx in TRPC6^−/− and WT MLEC. (Inset) Summary of Mn²⁺ influx experiments. (h–j) Representative current density–voltage relationships during normoxia and hypoxia, as well as during application of a membrane-permeable analogue of diacylglycerol (OAG, 100 μM) and of flufenamic acid, an activator of TRPC6 (Fluf., 100 μM), under normoxic conditions are displayed. (Insets) Summaries of current densities–voltage relationships at holding potentials of ±60 mV. (c) n=6–7 independent experiments. (d–f) n=56–172 cells. (g) n=9–10 cells. (h–j) n=6–10 cells. All statistical data were assessed using Student's t-test with Welsh's correction and are presented as mean±s.e.m.; *P<0.05. (h–j) n=6–10 cells. The data are presented as mean±s.e.m. Paired Student's t-test was used for comparison with normoxia. Unpaired Student's t-test was used for comparison with WT MLEC; ^*,#P<0.05.