Glutathione (gsh)

Carboxylated MNPs with a diameter of ~100 nm were ordered from Ruixi Biotech. Co., Ltd. (Xi’an, China). Free and FITC-labeled NA proteins were provided by Thermo Fisher Scientific (Shanghai, China). DNA, avidin and glutathione (GSH) were obtained from Sangon Biotech. Co., Ltd. (Shanghai, China). Beta-amyloid peptides, human serum protein (HSA), beta-secretase, bovine serum protein (BSA), 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC), N-hydroxysulfosuccinimide (NHS) and 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) were acquired from Sigma-Aldrich (Shanghai, China). The sequences of DNA are 5′-NH₂-TTT TTG TAA AAC GAC GGC CAG-3′ (capture probe DNA), 5′-TAG GAA TAG TTA TAA CTG GCC GTC GTT TTA C-3′ (target DNA, T_DNA) and 5′-TTA TAA CTA TTC CTA-biotin-3′ (bio-DNA). Monoclonal antibody (Ab₁) and biotinylated second antibody (bio-Ab₂) specific to Aβ₄₀ were obtained from Covance Inc. (Dedham, MA, USA). Aβ₁₆ was provided by China Peptide Co., Ltd. (Shanghai, China). Other reagents were ordered from Aladdin Reagent Co., Ltd. (Shanghai, China). The peptide of Aβ₄₀ was dissolved in HFIP solution and then reconstituted in NaOH to a concentration of 1 mM. Before its use, the peptide was diluted with 10 mM phosphate buffer (pH 7.2) to a desired concentration.

As detoxifying enzymes, tau class GSTs have many substrates. We chose the typical substrates for GST detoxification, including 1-chloro-2,4-dinitrobenzene (CDNB), 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl), 4-nitrobenzyl chloride (NBC), 1,2-dichloro-4-nitrobenzene (DCNB), and 4-nitrophenyl acetate (4-NPA) (Sigma-Aldrich Corp., St. Louis, MO, USA). For GOPX detoxification, we chose two substrates, cumene hydroperoxide (Cum-OOH) and ethacrynic acid (ECA) (Sigma-Aldrich), to test the effect of G- and H-site residues on substrate activity. The activities toward CDNB, NBC, DCNB, and ECA were measured as described by Habig et al. [33 (link)], the activity toward NBD-Cl was measured as described by Ricci et al. [34 (link)], and the activity toward Cum-OOH was measured as described by Edwards and Dixon [35 ]. The structures of substrates are shown in Table 1. All reactions were carried out at 25 °C using 60 mM GSH (Sangon Biotech). Each reaction was repeated three times, and experimental results were based on the mean values of three independent experiments.

hiPSC-CMs were intermittently exposed Lipopolysaccharide (LPS) (1 ug/ml, Sigma, St. Louis, MO,US) every 6 hours per day for 1 week, observing the long-term effects of oxidative stress on WT or PLEKHM2-KO hiPSC-CMs at day 40 post myocardial differentiation. The effect of inhibiting oxidative stress on PLEKHM2-KO hiPSC-CMs was examined by treating with glutathione (GSH) (2 mM, Sangon Biotech, Shanghai, China) for 10 days, and the culture medium with GSH was changed daily until day 40 post myocardial differentiation. Rapamycin (RAPA) (500 nM, Selleck Chemicals, Houston, TX, US) was used to inhibit the activation of the mTORC1 signaling pathway for 72 hours, and evaluating the effects of RAPA on autophagic flux, mitochondrial function, and myocardial contractility in WT or PLEKHM2-KO hiPSCs-CMS at D40 post myocardial differentiation.