Cell Culture, Reagents and Antibodies
Human prostate cancer PC-3 and LNCaP, as well as LNCaP sublines (LNCaP/Bclxl and LNCaP/LN11) were described in our previous publication [11]. Briefly, LNCaP/Bclxl cells were established by stable transfection of LNCaP cells with a vector bearing HA-tagged human bcl-xl cDNA sequence (pcDNA3.1-Bclxl.HA). LN11 is a LNCaP cell subline that lost Bcl-xL expression, as described [11]. Cells were maintained in a humidified atmosphere of 5% CO2, RPMI 1640 supplemented with 10% fetal bovine serum (FBS) with antibiotics (Invitrogen, Carlsbad, CA). Antibodies for PARP, caspase-3, CaSR and Actin were purchased from Santa Cruz Biotech (Santa Cruz, CA). CaSR small interference RNA (siRNA) mixture and the negative control siRNA were obtained from Santa Cruz Biotech. The calcimimetic R isomer of N-[3-[2-chlorophenyl]propyl]-[R]-α-methyl-3-methoxybenzylamine (NPS R-568) and its inactive isomer NPS S-568 were kindly provided by Amgen, Inc. (Thousand Oaks, CA).
Cell Viability Analyses
For MTT [3-[4,5-dimethylthazol-2-yl]-2,5-diphenyl tetrazolium-Bromide] assay, which is based on the conversion of MTT to MTT-formazan by mitochondrial enzyme, a cell growth determination kit (Sigma Co., St Louse, MO) was utilized according to the instruction from the manufacturer. Briefly, cells were seeded at a density of 2 × 103 cells/well in 96-well plates in triplicates and allowed to attachment overnight. Cells were then maintained in various conditions as indicated in the figures. The MTT solution was added in an amount equal to 10% of the culture volume. After 3 h incubation, the culture media was removed and the MTT solvent was added. The plates were read at a wavelength of 570 nM.
For trypan blue assay, cells were seeded in 12-well plates, and then treated with various reagents as indicated in the figures. At the end of experiments, viable cells was counted using a hemocytometer after staining with trypan blue as described in our recent publication [11].
For siRNA transfection, cells were plated in 6-well plates and transfected with the siRNA mixture as indicated in the figure using OligoTransfectamine™ (Invitrogen, Carlsbad, CA), as described in our previous publication [11]. Three days after transfection, cells were treated with the R568 at the concentrations indicated in the figure. Cellular survival was assessed with trypan blue exclusion assay.
To assess the cell death objectively, a LIVE/DEAD® Viability/Cytotoxicity kit (Invitrogen, Carlsbad, CA) was utilized. This kit provides two molecular probes, of which one probe labels the living cells as green based on an intracellular esterase activity and the other probe simultaneously labels the dead cells as red due to the disruption of plasma membrane integrity. The assay was conducted by following the protocol provided by the manufacturer. Briefly, cells were placed in 24-well plates overnight, and treated with R-568 for different time periods as indicated in the figures. At each time points, cells were incubated with the fluorescent dyes (2.0 μM) for 15 min before micro-images were taken under a fluorescent microscope.
Mitochondrial Membrane Potential (JC-1) assay
To examine the change of mitochondria membrane potential, JC-1 staining assay was used, as described in our previous publication [11]. Briefly, after treatment with R-568 or S-568 for 24 h, cells were incubated in the presence of JC-1 (Cell Technology Inc., Mountain View, CA) at a final concentration of 0.3 μg/ml for 15 minutes at 37C. Thereafter, the cells were analyzed under a fluorescent microscope.
Western Blot Analysis
Western blot was carried out as described previously [11]. Briefly, cells were pelletted and lysed in a buffer containing protease inhibitors (Half™ Protease Inhibitor Cocktail Kit, PIERCE, Rockford, IL). Equal amounts of proteins were separated on SDS-PAGE gels and transferred to PVDF membrane (BIO-RAD, Hercules, CA). Membranes were blocked in a Tris-buffered solution plus 0.1% Tween 20 (TBS-T) solution with 5% nonfat dry milk and incubated with primary antibodies overnight at 4C. Immunoreactive signals were detected by horseradish peroxidase-conjugated secondary antibodies and chemiluminescence substrate purchased from (Santa Cruz Biotech., Santa Cruz, CA).
Statistical Analysis
All cell culture-based experiments were repeated two or three times. Western blots are presented from representative experiments. The mean and SEM for cell viability assay are shown. The significant differences between groups were analyzed as described in our previous publication [11], using the SPSS computer software (SPSS Inc., Chicago, IL).