Thioredoxin reductase assay kit

Thioredoxin reductase activity was determined using a thioredoxin reductase assay kit (Abcam, Cambridge, MA, USA, Cat # ab83463). Briefly, HCT116 cells were treated with 0.2, 2, 4, 8, 10 μM of SIMR1281 for 48 h. Cells were lysed, and protein quantification was performed. After adjusting protein concentrations, two aliquots from the same sample were tested either with TrxR inhibitor or without it. Ten microliters of TrxR inhibitor was added to one set of the sample, and 10 μL of assay buffer was added to the other set. After adding the reaction mixture, OD was measured directly at 412 nm using a Varioskan Flash Multimode Spectral Scan Reader (Thermo Fisher Scientific, Waltham, MA, USA). OD was measured again after incubation for 20 min. Data analysis was performed according to the manufacturer’s instructions. Reduced glutathione (GSH) was quantified using a GSH/GSSG ratio detection assay kit (Abcam, # ab138881) as per the manufacturer’s instructions. After treating HCT116 cells with 0.4, 0.8, 1.5, 3, and 6 μM of SIMR1281 for 48 h, cells were harvested and lysed using 0.5% NP-40 lysis buffer. The TCA/NaHCO3 protein deproteinization step was performed, and the GSH assay mixture was applied to the samples. GSH was measured directly using a GSH standard; Fluorescence was monitored at Ex/Em = 490/520 nm using Multiskan go (Thermo Fisher Scientific, Waltham, MA, USA).

STM (purity > 98%) was purchased from Tauto Biotech (Shanghai, China). Dulbecco's Modified Eagle's Medium (DMEM) and fetal bovine serum (FBS) were obtained from Gibco (Eggenstein, Germany). Penicillin and streptomycin were purchased from Solarbio Co., Ltd. (Beijing, China). Annexin V-FITC apoptosis detection kit, ROS assay kit, mitochondrial membrane potential assay kit with JC-1, GSH/GSSG assay kit, and NAC were obtained from Beyotime (Nanjing, China). Propidium iodide (PI), calcein AM, dimethylsulfoxide (DMSO), protease inhibitor cocktail, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and phenylmethylsulfonyl fluoride (PMSF) were purchased from Sigma-Aldrich (St. Louis, MO). Thioredoxin reductase assay kit was obtained from Abcam. Human TNF-a was purchased from Sino Biological Technology (Beijing, China). The primary antibodies for Bax, Bcl-2, GAPDH, and Lamin B1 were obtained from Proteintech (Wuhan, China). Primary antibodies for cleaved caspase-3, cleaved PARP, and NF-κBp65 were obtained from Cell Signaling Technology (Beverly, MA). Horseradish peroxidase- (HRP-) conjugated secondary antibodies (goat anti-rabbit, goat anti-mouse) were obtained from Sigma.

Freshly collected leaves of the maize seedlings (Waza and Złota Karłowa cvs) were ground in liquid nitrogen. Total activity of the thioredoxin reductase (TrxR) was determined using the colorimetric Thioredoxin Reductase Assay Kit (Abcam, Cambridge, Great Britain; catalogue no. ab83463), following the manufacturer’s protocol. Portions of the powder (50 mg) were homogenized with 200 μL ice-cold Assay Buffer. Next, the samples were centrifuged at 10,000 × g for 15 min. (at 4 °C). The supernatant was collected and used for determination of TrxR activity. Measurement of TrxR activity was based on reduction of DTNB (5, 5′-dithiobis (2-nitrobenzoic) acid to TNB (5-thio-2-nitrobenzoic acid). The yellow color developed was measured at λ = 412 nm, using a microplate UV-Vis spectrophotometer (BioTek, Winooski, VT, USA). One unit (U) of TrxR activity was defined as the amount of enzyme that generates 1.0 μmol of TNB per minute at 25 °C. Total activity of TrxR in the maize samples was expressed as nmol of TNB per minute per milligram of protein. The protein content in the extracts was determined using Lowry method [54 (link)].