Pyridoxamine

Following incubation of hiPSCs with reactive carbonyl compounds for 24 h, the medium was removed and the cells were stained with Hoechst 33342 (1:2000, Thermo Fisher) in Dulbecco’s phosphate-buffered saline (DPBS). After 5 min of incubation, the staining solution was replaced, and the cells were washed with DPBS. Images were acquired via tile scanning using the Axio Observer (Zeiss, Jena, Germany). Cell number was automatically counted using the Zen software (Zeiss). We performed rescue experiments using pyridoxamine (Sigma Aldrich)^{33 (link)}. For hiPSC-derived neurons, cell viability was manually analyzed using the trypan blue exclusion test³⁴ . Briefly, the cell suspension was obtained from the cells treated with MGO for 24 h. Suspensions were gently mixed with 0.4% (w/v) trypan blue solution to detect viable cells. The number of live cells was counted using a hemocytometer (Wakenbtech, Kyoto, Japan).

Flash-frozen atria were thawed in 4 ml of phosphate-buffered saline (PBS) containing indomethacin 100 μmol/l (Sigma-Aldrich) to prevent formation of IsoLGs via oxygenation by cyclooxygenase of arachidonic acid released during the process, and pyridoxamine 1 mmol/l (Sigma-Aldrich) as an IsoLG scavenger. Tissues were homogenized using a jaw homogenizer and tissue grind tubes, before centrifugation at 10,000 × g for 20 min at 4°C. The supernatant was collected for protein IsoLG adducts analysis.
Cells subjected to stretch, and control cells simultaneously cultured on BioFlex plates, but without stretch, were incubated with indomethacin and pyridoxamine, in 1 ml of PBS (pH 7.4) at 4°C for 30 min before harvest.
Protein concentrations in homogenized atria or cells were measured using a BCA Protein Assay kit (Pierce, Rockford, Illinois), and samples were subjected to complete enzymatic digestion to individual amino acids (15 (link)). A [¹³C₆] internal standard was added, and the IsoLG-lysyl adducts were purified by solid-phase extraction and high-performance liquid chromatography before being quantified by liquid chromatography-tandem mass spectrometry assay using isotopic dilution as described previously (29 (link)).

The information for standards and reagents is as follows: methionine and cysteine were purchased from Jiuding Chemical Co., Ltd. (Suzhou, China). S-adenosylhomocysteine (SAH) was purchased from Leyan Reagent (Shanghai, China). Hcy, serine, and pyridoxamine were purchased from Sigma-Aldrich (St. Louis, MO, USA). Betaine, glutathione and taurine were purchased from Acmec Biochemical Co., Ltd. (Shanghai, China). The 5-MTHF was purchased from Yuanye Bio-Technology Co., Ltd. (Shanghai, China), and the purity of standards are all greater than 98%. Perfluoroheptanoic acid (PFHA) was purchased from Energy Chemical (Shanghai, China). Choline was purchased from CNW Technologies (Düsseldorf, Germany), and acetonitrile (Optima LC/MS grade) was purchased from F&H Co., Ltd. Ascorbic acid was purchased from Aladdin Co., Ltd. (Shanghai, China), methanol (mass spectrometry grade) was purchased from Fisher Company, and TCEP was purchased from Yuanye Bio-Technology Co., Ltd. (Shanghai, China). The experimental water was purified by the water purification system (18.2 MΩ, Sartorius, Göttingen, Germany). All the other reagents are at analytical grade.