Caspase activity assay kit

3-(4,5-Dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) was obtained from MP Biomedicals Inc. (Santa Ana, CA, USA). RPMI 1640 was purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Fetal bovine serum (FBS) were bought from Zhejiang Tianhang Biotechnology Co., Ltd. (Hangzhou, China). Anti-GAPDH antibodies were from Bioworld Technology Inc. (St. Louis Park, MN, USA), while anti-cytochrome c antibodies were acquired from Cell Signalling Technology Co. (Danvers, MA, USA). The caspase activity assay kit was a product of Beyotime Co. (Shanghai, China). Other routine laboratory reagents were obtained from commercial sources in analytical or HPLC grade. NMR data were recorded on a Inova-500 NB spectrometer (Varian, Palo Alto, CA, USA). CDCl₃ was used as solvent and TMS as internal standard. Chemical shifts (δ) are expressed in ppm with reference to the TMS peak. Mass spectra were acquired on an ultrahigh liquid chromatography instrument coupled with a quadrupole time-of-flight mass spectrometer (G6540A, UPLC-QTOF-MS, Agilent Technologies, Santa Clara, CA, USA). IR spectra were obtained on a 5DX-FTIR spectrophotometer (Nicolet, Madison, WI, USA). Column chromatography was performed on silica gel (200–300 mesh, Qingdao Marine Chemical, Qingdao, China). Fractions were monitored by TLC visualized by heating silica gel plates sprayed with 5% H₂SO₄ in EtOH.

Caspase activities were detected using a Caspase Activity Assay Kit (Beyotime). Briefly, cells were harvested, lysed, and centrifuged to collect the lysate supernatant. Subsequently, the total protein content was determined by the Bradford method. Finally, assay buffer, lysate supernatant, and Ac-DEVD-pNA were successively loaded on 96-well plates and incubated for 2 h at 37°C. The absorbance at 405 nm was detected by a microplate reader; then, the activity of caspase-3 was determined according to the pNA standard curve.

Caspase-3/8 activity assays were performed using a Caspase Activity Assay Kit (Beyotime Biotechnology, China). Briefly, the samples were lysed with lysis buffer for 15 min on ice. Then, the cells were centrifuged at 16,000 g for 20 min at 4°C, and the supernatant was immediately used for caspase-3/8 enzyme assays. Caspase activity was measured through the cleavage of a colorless substrate Ac-DEVD-pNA for caspase-3 and Ac-IETD-pNA for caspase-8, thus releasing pNA (p-nitroaniline). Absorbance was measured at OD_405 nm using a microplate reader (Thermo Scientific). The protein concentration of the cells was measured using a Bradford Protein Assay Kit (Beyotime Biotechnology, China). Caspases-3/8 activities were calculated using a standard curve. One unit was defined as the amount of enzyme cleaving 1.0 nmol of the colorimetric substrate pNA per hour at 37°C under saturated substrate concentrations. The assays described above were repeated five times.

Cells from each treatment group were harvested at 48 hours post‐transfection and caspase activity was measured using a caspase activity assay kit (Beyotime, Haimen, China). Cellular extracts and substrates (Ac‐DEVD‐pNA) were kept in 96‐well plate for 2 hours at 37°C. Absorbance values were measured using a microplate reader at 405 nm (Infinite M200, Tecan, Switzerland).