Gsh quantitation kit

SOD activity kit and GSH quantitation kit (Enzo Life Sciences Inc., Farmingdale, NY, USA) were used for evaluating antioxidant capability, according to manufacturer’s instructions. Briefly, to determine SOD activity, Master mix and xanthine solution were added to each well that containing protein extract or SOD buffer (control). SOD activity was determined by detecting WST-1 formazan (OD 450 nm; every one-min for ten times), which is conversed from WST-1 with xanthine oxidase. The activity was expressed as units per milligram of protein (U/mg protein). Total GSH level was quantitated by measuring the production of 5-thio-2-nitrobenzoic acid (yellow color) (OD 405 nm; every one-min for ten times) from GSH and DTNB. Briefly, samples were homogenized in 0.5 M perchloric acid and centrifuged at 12,000× g for 5 min. The supernatant was collected and neutralized (with 0.1 M phosphate buffer, pH 7.0, and 1 mM EDTA) for detection. Data were calculated by means of a calibration curve and normalized to the protein concentration.

2-iodo-4′-methoxychalcone (CHA79) was synthesized as previously described [15 (link)]. Chemical reagents, such as MG, hoechst 33342, 2′,7′-dichloro-dihydrofluorescein diacetate (H₂DCF-DA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), etc., were obtained from Sigma-Aldrich (St. Louis, MO, USA). Cell culture used materials were from Thermo Fisher Scientific (Waltham, MA, USA). Annexin V-FITC assay kit was from BD Bioscience (San Jose, CA, USA). SOD activity and GSH quantitation kit were from Enzo Life Sciences (Farmingdale, NY, USA). SDS-PAGE used materials were from Bio-Rad (Hercules, CA, USA). All primary and secondary antibodies used in Western blots (WB) are listed in supplemental material (Table S1).