Caspase 7

Anti-Bcl-2, caspase 3, caspase 7, cyclin B1, cyclin D1, CDK2, CDK4, CDC2, p21, p27, and β-actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and anti-PARP and phospho-p53 (Ser 15) antibodies were from Cell Signaling Technology (Danvers, MA, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and propidium iodide (PI) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Cisplatin (cis-diammine dichloroplatinum II; CDDP) was purchased from JW Pharmaceutical Co. (Seoul, Republic of Korea).

A human melanoma cell line (A375.S2) and a human epidermal keratinocyte cell line (HaCaT) were provided from one of co-author Feng-Lin Yen. Primary human fibroblasts were procured from the Food Industry Research and Development Institute (Hsinchu, Taiwan). Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12) was obtained from GIBCO (Grand Island, NY, USA) and supplemented with 10% fetal bovine serum (FBS) (Hazelton Research Products; Denver, PA, USA).Antibodies for Western blotting (phospho-p38, phospho-ERK, and phospho-JNK) were obtained from Cell Signaling Technology (Danvers, MA, USA). Puma, Bax, Bad, Bid, Apaf-1, cytochrome C, caspase-3, caspase-7, and caspase-9were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The GAPDH antibody was obtained from Biogenesis (Bournemouth, UK). N-acetylcysteine (NAC), apocynin (APO), U-0126, SB-202190, and SP600125 were procured from Biomol (Plymouth Meeting, PA, USA). MitoSOX Red mitochondrial superoxide indicator was purchased from Molecular Probes (Eugene, OR, USA). caspase-3, caspase-7, and caspase-9 colorimetric assay kits were obtained from R&D Systems (Minneapolis, MN, USA). Cudraflavone C was isolated from the methanolic extract of A. xanthocarpus as described previously in Reference [3 (link)], and its purity was determined (>97%) using high-performance liquid chromatography (HPLC) analysis.

Western blot assays were performed by fractionating either whole or cytoplasmic and nuclear proteins on PAGE-SDS gels followed by detection of NFATc1 using the 7A6 mAb or NFATc1/α Ab (IG-457). In addition, the expression of BCL6 (using Ab D65C10, Santa Cruz), c-Myc (9E10, Santa Cruz), caspase-3, caspase-7 (Santa Cruz) and NFATc2 (Santa Cruz) was investigated. In other assays, Namalwa cells were cultivated either with or without GaN treatment in the presence SYK inhibitor (P505-15) to detect p-AKT, AKT, p-mTOR, mTOR, and p-S6 proteins (all Abs from Santa Cruz). Signals were developed using a chemiluminescence detection system (Thermo Fisher Scientific).

Antibodies for western blotting were purchased from Cell Signaling (Danvers, MA): p-EGFR (Tyr1068) (2234, 1:1000), EGFR (4267, 1:1000), p-MEK1/2 (Ser217/221) (9154, 1:1000), MEK1/2 (8727, 1:1000), p-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (9101, 1:1000), p44/42 MAPK (Erk1/2) (9102, 1:1000), p-p38 MAPK (Thr180/Tyr182) (9211, 1:1000), p38 MAPK (9212, 1:1000), α-tubulin (2144, 1:2000), vimentin (3390, 1:1000), TACE (3976, 1:1000), Snail (3879, 1:1000), E-cadherin (3195, 1:1000), p-FAK (Tyr397) (3283, 1:1000), FAK (3285, 1:1000), p-STAT3 (Tyr705) (9131, 1:1000), STAT3 (9139, 1:1000), GAPDH (2118, 1:2000), β-actin (3700, 1:2000), p-DNA-PK (68716, 1:1000), DNA-PK (38168, 1:1000), PARP (9532, 1:1000), p-Histone H2A.X (Ser139) (9718, 1:1000), Histone H2A.X (2595, 1:1000), p-Chk2 (Thr68) (2197, 1:1000), Chk2 (2662, 1:1000), p53 (2524, 1:1000), caspase-7 (9492, 1:1000), caspase-3 (14220, 1:1000), AURK-A (14475, 1:1000); Santa Cruz Biotechnology (Dallas, TX): integrin β1/ITGB1 (sc-374429, 1:1000). Selumetinib (AZD6244, MEK1/2 inhibitor), osimertinib (AZD9291, EGFR^T790M), M3814 (DNA-PK inhibitor, DNA-PK-I) and ZM-447439 (AURK-A inhibitor, AURK-A-I), MK-4827 (PARP inhibitor, PARP-I), M4076 (ATM inhibitor, ATM-I), M6620 (ATM/ATR inhibitor, benzosertib) and M4344 (ATR inhibitor, ATR-I) were purchased from Selleck Chemicals (Selleckchem).

The western blotting assay of proteins extracted from the MCF7 cells treated with IC₅₀ concentration polycerasoidin was performed as previously described [24 (link)], with slight modifications. Briefly, protein separation (40 μg) was performed using 10% SDS-PAGE (20 mA, for 3 h). The separated proteins were transferred onto a PVDF membrane (Pierce, Rockford, IL, USA), which was subsequently blocked with Blocker Casein (Pierce) for 1 h at 25°C and then washed twice with TBST. The membranes were then incubated with the following primary antibodies at 4°C overnight: Bax (Cat: sc-493), Bcl-2 (Cat: sc-492), caspase-7 (Cat: sc-33773), caspase-9 (Cat: sc-7885), p21 (Cat: sc-397), p27 (Cat: sc-528) and β-actin (Cat: sc-7210) (1:1000; Santa Cruz Biotechnology, USA). This incubation was followed by a 1-h incubation with goat anti-rabbit and goat anti-mouse secondary antibodies conjugated to alkaline phosphatase (i-DNA, USA) at 25°C. After the membranes were washed twice with TBST, the bands were detected using the Fusion FX7 system (Vilber Lourmat, Germany).

CSF1, GM-CSF, IL-4, Q-VD-OPh and Z-VAD-FMK were obtained from R&D Systems (Minneapolis, MN, USA). Ac-YVAD-cmk from InvivoGen (San Diego, CA, USA), staurosporine and cleaved caspase-3 blocking peptide (#1050) from Cell Signaling (Danvers, MA, USA), IDN-6556 from MedChemtronica (Stockholm, Sweden), Tiron, TEMPO and Mito-TEMPO from Santa Cruz (Dallas, TX, USA). We used antibodies to calreticulin (#ab22683, Abcam, Cambridge, MA, USA), active caspase-3 (#9664, Cell Signaling), caspase-3 (#7148, Santa Cruz), caspase-3 (#9662, Cell Signaling), caspase-7 (#9492, Cell Signaling), caspase-7 (#sc-81654, Santa Cruz), caspase-8 (#9746, Cell Signaling), caspase-8 (#AF705, R&D Systems), DIABLO (#2409, ProSci, Poway, CA, USA), FADD (#2782, Cell Signaling), GST (#sc-138, Santa Cruz), γ-H2AX (#05-636, Merck Millipore, Billerica, MA, USA), HSC70 (#ADI-SPA-816, Enzo Life Sciences, Farmingdale, NY, USA), mtHSP70 (#MA3-028, Thermo Fisher Scientific, Waltham, MA, USA), LC3 (#M152-3, MBL International, Woburn, MA, USA), NOX2 (#sc-130543, Santa Cruz), p47^PHOX (#4312, Cell Signaling), TOM20 (#sc-11415, Santa Cruz).