Y-27632 br Mitochondrial membrane potential br Alteration of
Mitochondrial membrane potential
Alteration of mitochondrial membrane potential (DCm) was measured using a fluorescent probe JC-1 dye (tetraethylbenzimi-dazolylcarbocyanine iodide). The MDA-MB-231 cells were seeded in 6-well plates at a density of 2 105 cells/well and incubated for 24 h. Cells were treated with DMSO, nor-wogonin (10, 20 or 40 mM), or 25 mM FCCP (positive control) for 24 h. and incubated in a CO2 incubator at 37 C. The cells were then stained with JC-1 reagent (100 ml) for 30 min. at 37 C. Mitochondria with normal function (normal DCm) emits red fluorescence whereas the
depolarized mitochondria emits green fluorescence. Red fluores-cence (excitation 550 nm, emission at 600 nm) and green fluorescence (excitation 485 nm, emission at 535 nm) intensity were measured using fluorescence plate reader. The ratio of
fluorescent intensity of red J-aggregates to fluorescent intensity of green J-monomers was used as indicator of the loss of DCm. The lower the value of this ratio, the lower the mitochondrial membrane potential.
Fig. 2. Effects of nor-wogonin on Y-27632 distribution and cell cycle regulatory proteins in MDA-MB-231 cells. A. Percentage of cells in the sub G1, G1, S, and G2/M phases after 24-h incubation with DMSO or 10, 20, or 40 mM nor-wogonin. B. Percentage of cells in the sub G1, G1, S, and G2/M phases after 24, 48, and 72-h incubation with DMSO or 30 mM nor-wogonin. C. Western blot analyses for determination of the expression of cell cycle regulatory proteins (p21, cyclin D1, CDK4, cyclin B1, and CDK1) following 24-h treatment with nor-wogonin (10–40 mM), compared to the negative control (DMSO) D. Western blot analyses for determination of the expression of cell cycle regulatory proteins (p21, cyclin D1, CDK4, cyclin B1, and CDK1) following 24, 48, or 72-h treatment with nor-wogonin (30 mM), compared to the negative control (DMSO). E. The protein expression levels of p21, cyclin D1, CDK4, cyclin B1, and CDK1 relative to GAPDH, following 24 h treatment with nor-wogonin (10–40 mM) were quantified using ImageJ software. F. The protein expression levels of p21, cyclin D1, CDK4, cyclin B1, and CDK1 relative to GAPDH, following 24, 48, and 72-h treatment with nor-wogonin (30 mM) were quantified using ImageJ software Values represent the means SD of three independent experiments. *p < 0.05 indicate significant differences, compared to the DMSO-treated control.
Western blot analyses
MDA-MB-231 cells were incubated with nor-wogonin, nor-wogonin combined with 30 mM pan-caspase inhibitor (Z-VAD-FMK), or DMSO for 24 h. For the time course experiment, MDA-MB-231 cells were incubated with nor-wogonin (30 mM) for 24, 48, or 72 h. MCF-10A cells were incubated with nor-wogonin (40 mM) or DMSO for 24 h. To generate whole cell lysate (WCL), cells were harvested, washed twice with chilled PBS, and lysed with an ice-cold lysis buffer containing 0.1% sodium dodecyl sulfate (SDS), 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 1% Triton X-100, 2 mg/ml aprotinin, 5 mg/ml 4-(2-aminoethyl)benze-nesulfonyl fluoride hydrochloride (Pefabloc SC; a protease inhibitor cocktail), 1% phosphatase inhibitor cocktail, and 50 mM Tris HCl (pH 7.4). The cell lysates were kept on ice for 30 min, collected, and centrifuged at 14,000 g for 15 min at 4 C. To generate nuclear protein extract for p65 assessment, cells were suspended in 420 ml of buffer A (10 mm HEPES, pH 7.9, 10 mm KCl, 0.1 mm EDTA, 0.1 mm EGTA, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, 20 mm β-glycerophosphate, 0.1 mm sodium orthovanadate, 10 mg/ml aprotinin, and 10 mg/ml leupeptin) and chilled on ice for 15 min. Then, 25 ml of 10% Nonidet P-40 was added, and the suspension was vortexed, and centrifuged at 15,000 rpm for 5 min. The nuclear pellets were washed with 200 ml of buffer A and suspended in 50–100 ml of buffer B (20 mm HEPES, pH 7.9, 0.4 m NaCl, 1 mm EDTA, 1 mm EGTA, 1 mm dithiothreitol, 1 mm phenyl-methylsulfonyl fluoride, 20 mm β-glycerophosphate, 1 mm sodium orthovanadate, 10 mg/ml aprotinin, and 10 mg/ml leupeptin). The mixture was kept on ice for 15 min with frequent agitation. Nuclear extracts were prepared by centrifugation at 15,000 rpm for 5 min and stored at 80 C. The protein concentration was determined by the Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA), according to the manufacturer’s instructions. For western blot analysis, 30 mgof protein was loaded onto a 10% or 15% SDS-PAGE gel according to the molecular weight of the tested protein. Proteins separated on the SDS-PAGE gel were transferred to a polyvinylidene fluoride (PVDF) membrane. The PVDF membrane was incubated in a blocking buffer containing 3% non-fat milk powder, 1% BSA (Sigma-Aldrich), and 0.5% Tween-20 in PBS for 1 h. Subsequently, the PVDF membrane was incubated with the suitable and validated primary antibody (Cell Signaling Technology, Danvers, MA, USA) overnight, followed by horseradish peroxidase (HRP)-conjugated IgG (Cell Signaling Technology, Beverly, MA, USA) for 1 h with gentle agitation at room temperature. Signals were detected by enhanced chemiluminescence (ECL) prime (GE Healthcare, Little Chalfont, UK) and autoradiography using an X-ray film (Konica Minolta Medical Imaging, Wayne, NJ, USA)