Cleaved parp

Curcumin (CAS Number 458-37-7, Sigma-Aldrich, Purity: ≧99.5%), demethoxyCurcumin (CAS Number: 22608-11-3, Sigma-Aldrich, Purity: ≧98%), bisdemethoxyCurcumin (CAS Number: 33171-05-0, Sigma-Aldrich, Purity: ≧98%), Thiazolyl Blue Tetrazolium Bromide (MTT) were purchased from Sigma–Aldrich (CAS Number: 298-93-1). β-actin (Cat No. GTX109639), p-p38 (Cat No. GTX24822), p-Akt (Cat No. GTX128414), p-Erk (Cat No. GTX24819), p-smad2 (Cat No. GTX133475), p-smad3 (Cat No. GTX129841), cleaved PARP (Cat No. GTX132329), cleaved caspase 3 (Cat No. GTX86952) antibodies were purchased from GeneTex. Annexin V Alexa Fluor 488 Ready Flow Conjugate (Lot number 2081235) and 7-aminoactinomycin D (7-AAD, Catalog number: A1310) both were purchased from Thermo Fisher Scientific.

After treatment with CGA, EGCG, and TC-HT for 24 h alone or in combination, cells were harvested, washed with cold PBS, and lysed on ice for 30 min in lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.25% deoxycholic acid, 1% NP-40, 1.0% Triton X-100, 0.1% SDS, 1 mM EDTA, 1% phosphate and protease inhibitor cocktail) (Millipore). Cell lysates were clarified by centrifugation at 23,000 × g for 30 min at 4°C, and the protein concentration in the supernatant fraction was quantified using the Bradford protein assay (Bioshop). Proteins were resolved by 10% SDS-PAGE and electrotransferred onto polyvinylidene fluoride membrane (PVDF) (Millipore) in transfer buffer (10 mM CAPS, pH 11.0, 10% methanol). The membranes were blocked with 5% nonfat dry milk/TBST (blocking buffer) for 1 h at room temperature and then incubated overnight at 4°C with diluted primary antibodies in blocking buffer. The specific primary antibodies against Bcl-2, cleaved caspase-8, cleaved caspase-9, cleaved caspase-3 (Cell Signaling), Bax (Santa Cruz), Cdc2, cyclin B1, cleaved PARP and β-actin (GeneTex) were used. After washing with TBST, the membranes were incubated with HRP-conjugated anti-goat (GeneTex) or anti-rabbit (Jackson Immunoresearch) secondary antibody. Chemiluminescence was detected using WesternBright ECL western blotting reagent (Advansta).