Mitosox

The intracellular total and mitochondrial ROS level were measured respectively by 2’,7’-dichlorofluorescin diacetate (DCFH-DA, S0033; Beyotime, Beijing, China) and MitoSOX (40778ES50; Yeasen biotech, Shanghai, China) staining. Both of them can be rapidly oxidized to highly fluorescent compounds. The samples were stained according to the manufacturers’ instructions and detected by FACSCalibur flow cytometer (BD Biosciences). Further, the mitochondrial ROS was evaluated by the colocalization of MitoSOX and Mitotracker (C1048, Beyotime) intensity detected using Laser Scanning Microscope (ZEISS LSM780, Germany).
MMP was detected using JC-1 (C2006; Beyotime) assay kits as described previously [30] (link) and analyzed by FACSCalibur flow cytometer (BD Biosciences). The decrease ratio of red (JC-1 aggregates) to green (JC-1 monomers) indicated the MMP loss. The mPTP Assay Kit (GMS10095.1; Genmed, Shanghai, China) was used to detect mPTP activity, wherein fluorescence quenching by cobalt ions increased when the mPTP activity increased. NP cell staining and fixation were performed according to the manufacturer's instructions. Next, the slides were observed and imaged using a fluorescence microscope (Olympus IX71).

Relative levels of ROS were determined using a fluorometric method, the dichloro-dihydro-fluorescein diacetate assay (DCFH-DA; Beyotime). Because free radical generation is an early event, ROS and superoxide were detected at 2 hours, as previously reported (Wang et al., 2015; Fu et al., 2016). Briefly, cells were seeded at 1 × 10⁴ cells/well in a 96-well plate, then incubated for 1 hour with or without 20–80 µM cyanidin, and co-incubated with 10 µM Aβ_25–35 for 2 hours. DCFH-DA (10 µM) was added to the cells, and ROS generation was monitored at excitation and emission wavelengths of 488 nm and 525 nm, respectively. The superoxide was detected by a mitochondria-targeted fluorogenic dye, MitoSOX (Beyotime).

Ginsenoside Rg1 was obtained from Nanjin Jingzhu Biotechnology Company. Assay kits for Cell Counting Kit-8, NO nitrate reductase, dihydroethidium (DHE), MDA, JC-1, and MitoSOX were obtained from Beyotime Biotechnology (Shanghai, China). Antibodies against calpain-1 and β-actin were obtained from Proteintech (Wuhan, China). Antibodies against Drp1, Mfn2, PP2A, Ser¹¹⁷⁷ eNOS phosphorylation and endothelial nitric oxide (eNOS) were obtained from ABclonal (Wuhan, China). Phenylephrine (PE), acetylcholine (Ach), polyethylene glycol-catalase (PEG-Cat), 5-hydroxydecanoate (5-HD) and okadaic acid (OKA) were obtained from Sigma Aldrich (Shanghai, China). MDL-28170, L-NAME, acetylcysteine (N-acetyl-l-cysteine, NAC), TEMPOL and rotenone were obtained from Selleck (Houston, TX, USA).

Doxorubicin hydrochloride (DOX·HCl) was purchased from Meilun Biological Technology Co., Ltd (Liaoning, China). Triethylamine (TEA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), Hoechst 33258, dimethyl sulfoxide (DMSO) and BCA protein assay kit were purchased from Sigma-Aldrich Co. (St Louis, MO, USA). Annexin V-FITC apoptosis detection kit was purchased from Sino Biological, Inc. (Beijing, China). The detection kits of ATP, ROS, Mito-sox and JC-1, were purchased from Beyotime Institute of Biotechnology (Shanghai, China). Unless stated otherwise, the other reagents were purchased from Sigma-Aldrich.
Culture medium (RPIM 1640 medium), FBS were purchased from Gibco Life Technologies (Thermo Fisher Scientific, Waltham, MA, USA). Mouse anti-GAPDH antibody, rabbit anti-Bcl-2 antibody and rabbit anti-Bax antibody were purchased from Abcam (Burlingame, CA, USA). Cleaved caspase-3 rabbit mAb was purchased from Cell Signaling Technology Inc. (Beverly, MA, USA). Goat anti-mouse IgG and goat anti-rabbit IgG were purchased from Beijing Biosynthesis Biotechnology Co., Ltd (Beijing, China).