Caspase 3 assay kit

N-CUR was obtained from Lipolife (Essex, UK) and DFO was supplied by Novartis Pharma AG (Rotkreuz, Switzerland). CuSO₄, CUR, primers, carboxymethylcellulose (CMC), sodium dodecyl sulfate (SDS), agarose, pyrogallol, thiobarbituric acid (TBA), and reduced glutathione (GSH) were obtained from Sigma (St. Louis, MO, USA). Caspase-3 assay kit, NF-κB p65, cardiac troponin I (cTnI), Bax and Bcl-2 ELISA kits were supplied by MyBiosource (San Diego, CA, USA), and TNF-α and IL-6 ELISA kits were supplied by R&D Systems (Minneapolis, MN, USA). Creatine kinase (CK)-MB and lactate dehydrogenase (LDH) kits were purchased from Spinreact (Girona, Spain). Antibodies against TLR4, p-p38 MAPK, p38 MAPK, pJNK, JNK, pERK1/2, ERK1/2, and β-actin were supplied by Novus Biologicals (Centennial, CO, USA). Other chemicals and kits were supplied by standard manufacturers.

The caspase-3 assay was used to determine the apoptotic effect of WSR extract and cisplatin in parent and drug resistance cell lines. EMT6/P flasks were treated with IC₅₀ concentrations of WSR extract (2.9 mg/ml), cisplatin (positive control; 20 μM), and combination treatment of the extract and cisplatin (0.54 mg/ml + 10 μM, respectively). EMT6/CPR flasks were treated with IC₅₀ concentrations of the extract (3.8 mg/ml), cisplatin (positive control; 54 μM), and combination treatment of the extract and cisplatin (0.66 mg/ml + 27 μM, respectively). MEM was used as the negative control. The flasks were then incubated for 48 h. After treatment, cells were collected, washed, and lysed using lysis buffer. Caspase-3 activity was measured using the procedure provided in the standard kit (Caspase-3 Assay Kit, My BioSource, United States).