Ac devd pna

The activity of caspase-3 enzymes was measured in cell lysates by a colorimetric assay kit (CASP-3-C, SIGMA-ALDRICH, Saint Louis, MO, USA), that is based on the hydrolysis of the compound acetyl-Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVD-pNA) (A 2559, Sigma-Aldrich, Saint Louis, MO, USA) by caspase-3. The reaction releases the p-nitroaniline moiety (p-NA) that absorbs at 405 nm. Cells were pre-incubated with 5 μΜ doxorubicin hydrochloride (DOX) (Sigma–Aldrich, Deisenhofen, Germany) for 3 h for caspase cascade initiation [41 (link),42 (link)]. Samples (10 μL) were tested both with and without the caspase-3 inhibitor acetyl-Asp-Glu-Val-Asp-al (Ac-DEVD-CHO, 20 μΜ final concentration), in a total reaction volume of 100 μL in 96-well plates. The substrate Ac-DEVD-pNA concentration was 200 μM and the assay was performed at 37 °C for 90 min. The caspase-3 specific activity was expressed as the percentage of μmol of p-nitroaniline released per min per μg of total protein values compared to control.

MTT (3-(4, 5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide), streptomycin, and penicillin were obtained from Amresco (Solon, OH). BSA, PMSF, and Na₃VO₄ were provided by Beyotime (Nantong, China). NADPH was obtained from Roche (Mannheim, Germany). The Annexin V-FITC and PI apoptosis assay kit was purchased from Zoman Biotech (Beijing, China). DMEM, puromycin, DMSO, G418, DCFH-DA, DHE, DTT, Hoechst 33342, Ac-DEVD-pNA, insulin, and CHAPS were products of Sigma-Aldrich (St. Louis, United States). FBS was a product of Sijiqing (Hangzhou, China). DTNB was a product of J&K Scientific (Beijing, China). All other reagents used were of analytical purity. The recombinant rat TrxR1 (WT TrxR1) was provided by Professor Jianqiang Xu (Dalian University of Technology, China). The Escherichia coli (E. coli) Trx was produced as described by our previous reference (Liu et al., 2014 (link)). The plasmids, shTrxR1 and shNT, and the cell lines, HEK-TrxR1 cells and HEK-IRES cells were provided by Prof. Constantinos Koumenis of the University of Pennsylvania, United States (Nalvarte et al., 2004 (link); Javvadi et al., 2010 (link)). The establishment of HeLa-shTrxR1 cells and HeLa-shNT cell lines refer to our previously published literature (Duan et al., 2014 (link)).

Caspase-3 activity was used as a marker for cell apoptosis and was determined according to Nicholson et al. [30 (link)]. The enzyme activity was measured with the Ac-DEVD-pNA colorimetric assay as previously described [31 (link)]. Briefly, cultured cells were lysed on ice with a lysis buffer (50 nM HEPES, pH 7.4, 100 nM NaCl, 0.1 % CHAPS, 1 nM EDTA, 10 % glycerol, and 10 nM DTT). The reaction was initiated by the addition of caspase-3 substrate Ac-DEVD-pNA (N-acetyl-Asp-Glu-Val-Asp-p-nitroanilide; Sigma) to a final concentration of 200 µM. The samples were incubated in the dark at 37 °C for 1 h. Then, the colorimetric release of p-nitroaniline from the Ac-DEVD-pNA substrate was recorded every 30 min and monitored continuously for 120 min at 405 nm in a microplate reader (Bio-Tek ELx800). Cells that were treated with 1 nM staurosporine were used as a positive control (Sigma, St Louis, MO, USA). The data were analyzed with KCJunior (Bio-Tek Instruments), normalized to the absorbance in vehicle-treated cells, and expressed as the percent of control ± SEM of ten separate samples that were run in triplicate. The absorbance of the blanks, acting as no-enzyme controls, was subtracted from each value. Because the initial substrate concentration was subsaturating (200 µM), only the data within the linear range of the reaction curve provided a consistent measure of caspase-3 activity.

Caspase-3 activity was assayed using a caspase-3 colorimetric activity assay kit (BIOMOL International, PL, USA). This study is based on a spectrophotometric assay of p-nitroaniline (p-NA) once the cleavage of caspase-3 substrate, Ac-DEVD-pNA (Sigma, St. Louis, MO, USA). About 3 × 10⁶ A549 cells were exposed to OEO (½ IC₅₀ and IC₅₀) and Etoposide (IC₅₀) for 4 h. After harvesting the cells and washing with cold PBS, the cells were lysed by cell lysis buffer [PIPES (20 mM), Mgcl (2 mM), KCl (10 mM), DTT (4 mM), EGTA (1 mM), EDTA (2 mM), containing PMSF (1 mM), pepstatin (1 mM), leupeptin (2.2 μM), and benzamide chloride (0.5 mM)] on ice. The cell lysates were centrifuged (12,000g, 4 °C), and then the supernatants were used to estimate the protein concentration using the Bradford method. In the following, equal amounts of each protein (100 μg), the colorimetric caspase-3 substrate (AcDEVD-pNA, 5 μL of 2 mmol/l) and assay buffer [Nacl (100 mM), HEPES (50 mM), DTT (10 mM), CHAPS (0.1%), EDTA (0.1 mM), Glycerol (10%)] were mixed, and the reaction mixtures were incubated (at 37 °C in darkness for 3 h) and the absorbance was measured at 405 nm using a microplate reader^{68 (link)}.