Mitosox

Mito-TEMPO and MitoSOX were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against CHOP, BiP, GRP94, β-actin, and HRP-conjugated secondary antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against SOD2 and caspase-12 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against Sirt3 and acetyl-K68-SOD2 were obtained from Abcam (Cambridge, MA, USA). T-SOD assay kit, SOD2 assay kit, MDA assay kit, ATP assay kit, and mitochondrial isolation kit were purchased from Beyotime Institute of Biotechnology (Jiangsu, China).

Intracellular ROS was measured by MitoSOX (Thermo Fisher Scientific; Rockford, IL, USA) and 2’,7’-dichlorofluorescein diacetate (DCF) (Sigma, St Louis, MO, USA) as previously described.[21 (link)] At the end of reoxygenation, the cardiomyocytes were stained with 5 μM MitoSOX Red or 5 μM DCF to detect superoxide anion. Cells were washed and imaged using a Nikon-Eclipse80i confocal microscope with 561 nm and 488 nm excitation for MitoSOX Red and DCF respectively. The fluorescence intensity of MitoSOX Red and DCF were qualified with ImageJ software, as reported previously.[22 (link)] The data are presented as fold change in the median intensity of the fluorescence when compared with the respective controls.

Measurement of superoxide in cells was performed as previously described11 (link). Briefly, cells were seeded onto coverslips placed at the bottom of 6-well plates and treated as detailed above. MitoSOX™ and bis Benzimide 33342 trihydrochloride (Hoechst; Sigma; used for counter stain) were added to the culture media for 10 min, at a final concentrations of 5 μM and 0.2 μg/mL, respectively. Following incubation with MitoSOX and Hoechst, cells were rinsed in PBS and fluorescence was visualized (excitation/emission at 510/580 nm).

For quantifying the production of mitochondrial O₂^·−, ECs were incubated with Mitosox Red (0.5 μmol/L; Life Technologies Inc.) for 30 minutes in the dark. The fluorescence intensity was measured with a microtiter plate fluorimeter (Enspire Perkin Elmer) and each reading was normalized to protein concentration. Data were expressed as fold increase over control. Additionally, ECs plated in coverslips were incubated with Mitosox, counterstained with DAPI (Sigma) and visualized with a confocal microscope (Leica TCS SP2, 40× objective), λ_excitation = 510 and λ_emission = 580 nm, using the same imaging settings in each case.

Ginkgo Biloba extract (GBE), prepared from Ginkgo biloba dropping pills, was kindly provided by Wanbangde Pharmaceutical Group Co., Ltd. (Wenling, China). Chemicals such as MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), DAPI (dihydrochloride), mitoSOX, rhodamine123, NSC23766, VAS2870 and DMSO (dimethyl sulfoxide) were obtained from Sigma-Aldrich (St. Louis, MO, USA), ThermoFisher (Waltham, MA, USA) and MedChemExpress (Shanghai, China). The following antibodies were obtained from Santa Cruz Biotechnology (Dallas, CA, USA) or Abcam (Cambridge, MA, USA): Bax (cat. no. ab32503), Bcl-2 (cat. no. ab196495), Caspase-3 (cat. no. ab184787), CAV-1 (cat. no. ab32577), NOX2 (cat. no. sc-130543), p-SRC (cat. no. ab40660), SRC (cat. no. ab133283), p-Vav2 (cat. no. ab86695), Vav2 (cat. no. ab52640), Rac1 (cat. no. ab155938) and GAPDH (cat. no. sc-365062). The Rac1 activity assay kit (cat. no. STA-401-1) was obtained from Cell Biolabs (San Diego, CA, USA). Other chemicals and reagents used in the present study, unless otherwise specified, were obtained from Beyotime (Nantong, China) and Sangon (Shanghai, China).