Caspase 3

Cells were lysed in modified RIPA buffer containing 150 mM NaCl, 1% NP-40, 50 mM Tris-HCl (pH 8), 0.5% deoxycholic acid, 0.1% SDS, 1% Triton X-100, protease and phosphatase inhibitors. Lysates were placed on ice for 45 minutes and then clarified by centrifugation. Supernatants were removed and total protein measured by Bradford assay. Forty microgram of protein lysate per lane was electrophoresed on 12% SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked for 1 h in TBST blocking solution, containing 5% bovine serum albumin and then incubated with a primary antibody overnight at 4 °C. The membranes were washed at least 3 times with each wash for 10 min with washing solution (TBS and 0.1% Tween 20) and incubated for 45 min with appropriate horseradish peroxidase-conjugated secondary antibodies. The washed membranes were developed using ECL Blotting Substrate (Thermo Scientific). The β-actin,VDR, ORF57, K8α and ERK antibodies were purchased from Santa Cruz Biotechnology. The phospho-p38 mitogen-activated protein kinase (MAPK), LANA and caspase-3 antibodies were purchased from Imgenex.

MPT0E028 and SAHA were synthesized by Professor Jing-Ping Liou. RPMI-1640 medium, FBS, penicillin, streptomycin, and all other tissue culture reagents were obtained from GIBCO/BRL Life Technologies (Grand Island, NY, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), propidium iodide (PI), LY294002 and all of the other chemical reagents were purchased from Sigma Chemical (St. Louis, MO, USA). The following antibodies were used: caspase 8, caspase 9, p-Akt(T308), Akt, p-mTOR, mTOR, p-GSK3β, p-eIF4E, p-p70S6K(T421/S424), p-p70S6K(T389), HDAC1, HDAC2, HDAC4, acetyl-α-tubulin, BID, STAT2, STAT4, STAT5A, STAT6 (Cell Signaling Technologies, Boston, MA, USA); PARP, HDAC6, HRP-conjugated anti-mouse and anti-rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA); p-Akt (S473) (Epitomics, Burlingame, CA, USA); caspase 6, caspase 7, p53, STAT1 (BD Biosciences PharMingen, San Jose, CA, USA); caspase 3 (Imgenex, San diego, CA, USA); acetyl-histone H3, STAT5B (Upstate Biotechnology, Lake Placid, NY, USA); Actin (Chemicon, Billerica, MA, USA). Trizol reagent was from Invitrogen (Carlsbad, CA, USA). Random primer and M-MLRT were purchased from Promega (Madison, WI, USA). Pro-Teq was from Protech (Taipei, Taiwan).

Vincristine and suberoylanilide hydroxamic acid (SAHA) were purchased from Sigma Chemical Co. (St. Louis, MO, USA), and tubastatin A (HDAC6 inhibitor) was synthesized from Dr. Jing-Ping Liou (Taipei Medical University, Taiwan). The above drugs were dissolved in DMSO (dimethylsulfoxide) and then preserved at −20 °C. Rhodamine 123, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium, propidium iodide, anti-β-tubulin, FITC-conjugated anti-mouse IgG, and all of the other chemical reagents used in this study were purchased from Sigma Chemical (St. Louis, MO, USA) and of analytical grade. Primary antibodies against Cdc2 (pY15), aurora B, caspase 8, caspase 9, HDAC1, HDAC2, HDAC3, HDAC4, and Bid were all purchased from Cell Signaling Technology (Beverly, MA); cyclin B, Cdc25C, Cdc2, PARP, Mcl-1, Bcl-2, Bcl-xl, and secondary antibodies were purchased from Santa Cruz (Santa Cruz, CA, USA); MPM2 (pSer/Thr) and H3 (pS10) were purchased from Upstate Biotechnology (Lake Placid, NY, USA); PLK (pT210), caspase 6, and caspase 7 were purchased from BD Biosciences (San Jose, CA, USA); caspase 3 was purchased from Imgenex (San Diego, CA, USA); acetyl-histone 3 was purchase from Millipore (Billerica, MA, USA); and an internal control, GAPDH, was purchased from Novus Biologicals (Littleton, CO, USA). Vectashield® mounting medium with DAPI was purchased from Burlingame, CA, USA.

Lanatoside C (lana.C), rottlerin, Ro318220, Gö6983 were purchased from Sigma Chemical Co. (St Louis, MO, USA). The above drugs were all dissolved in dimethylsulfoxide (DMSO). The non-conjugated primary antibodies were against C23 (nucleolin, Sigma Chemical Co., St Louis, USA), BID, caspase-8, caspase-9, PKCδ (Thr505), PKCδ, caveolin, phoshpo-p44/42 MAP kinas (Thr202/Tyr204), p44/42 MAP kinas (Thr202/Tyr204), phosho-SAPK/JNK (Thr183/Tyr185), p38 MAP kinase, phosho-mTOR, phosphor-p70 S6 kinase (Thr421/Ser424), phosphor-4E-BP1 (Thr37/46), phosho-eIF4E (Ser209), AKT (Cell signaling Technology, Beverly, MA, USA), AIF, Bcl-xl, Mcl-1, α-tubulin, PARP-1/2 (Santa Cruz Biotechnology Corp., Santa Cruz, CA, USA), caspase-2, caspase-6, caspase-7 (BD Biosciences, San Jose, CA, USA), phosho-AKT (Ser473, Epitomics Inc., Burlingame, CA, USA), caspase-3 (Imgenex, San Diego, CA, USA), β-actin (Millipore, Temecula, CA, USA). The secondary horseradish peroxidase (HRP)-conjugated anti-mouse and anti-rabbit IgGs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). z-VAD-fmk was purchased from R&D system (Minneapolis, MN, USA).

Human prostate adenocarcinoma cell lines, PC-3 and DU-145, were obtained from American Type Culture Collection (Rockville, MD, USA). RPMI 1640 medium, fetal bovine serum (FBS), penicillin, and streptomycin were purchased from GIBCO/BRL Life Technologies (Grand Island, NY). Antibodies of PARP-1, Bcl-2, Bcl-xL, Bak, Mcl-1, α-tubulin, cyclin A, cyclin B, cyclin-dependent kinase (Cdk) 1, and GAPDH were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies of cleaved caspase-9, caspase-8, β-tubulin (Alexa Fluor 594 Conjugate), p-Cdk1^Thr161, and p-Cdk1^Tyr15 were from Cell Signaling Technologies (Boston, MA). Stathmin-1, BUBR1, and CENP-A were from Abcam (Cambridge, UK). MPM2 was from Millipore (Bedford, MA, USA). Caspase-3 was purchased from Imgenex (San Diego, CA). Antibody of PDE5 was from OriGene Technologies (Rockville, MD, USA). PDE5 small interfering RNA (siRNA) was from GE Healthcare Dharmacon (Chicago, USA). JC-1 and DAPI were from Molecular Probes (Eugene, OR, USA). Anti-mouse and anti-rabbit IgGs were from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). Leupeptin, phosphatase inhibitors (NaF and Na₃VO₄), dithiothreitol, phenylmethylsulfonylfluoride (PMSF), propidium iodide (PI), and all other chemical compounds were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Total protein isolated from cultured cell lines or tumor tissues from xenograft animal studies were lysed in modified RIPA buffer (50 mM Tris-HCl at pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.25% sodium deoxycholate) with 1 mM DTT, 10 mM NaF, 1 mM PMSF, 1 μg/ml of aprotinin, 1 μg/ml of leupeptin, 1 mM Na₃VO₄, and the phosphatase inhibitor cocktail to be analyzed. Following lysis, the lysates were resolved on an SDS-containing 10% polyacrylamide gel were transferred to a polyvinylidene difluoride nylon (PVDF) membrane and were probed with specific antibodies. The specific bands were detected by horseradish peroxidase-conjugated antibody and were revealed by Western Lighting^® Plus-ECL (PerkinElmer, Waltham, MA) and X-ray film (Fujifilm, Tokyo, JP). The antibodies used were PARP-1 (F2), phospho-histone H3 and GAPDH from Santa Cruz Biotechnology (Santa Cruz, CA), Aurora-A [35C1] from GeneTex (Irvine, CA), and caspase-3 from IMGENEX (San Diego, CA).