Cell culture and pharmacological intervention
The human prostate cancer PC-3 cells (obtained from Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center of Cancer, Tianjin, China) were cultured in RPMI 1640 medium (Gibco, CA, USA) with 10% (v/v) fetal bovine serum (Gibco, CA, USA) containing antibiotics (100 U/mL penicillin G and 100 mg/mL streptomycin) and incubated at 37˚C in 5% CO2 humidified incubator. As per experimental requirements, PC-3 cells were treated with valproic acid (VPA, purchased from Sigma-Aldrich Chemicals, MO, USA) and then subjected to 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assay, wound-healing assay, Oil-Red O staining, 4′,6′-diamidino-2-phenyl-indole (DAPI) staining assay, Annexin V-FITC and propidium iodide (PI) double-staining assay, quantitative real-time polymerase chain reaction (qRT-PCR), and Western blotting analysis.
MTT cell viability assay
PC-3 cells were seeded in a 96-well plate at a density of 2 × 104 cells/well and incubated overnight, followed by treatment with the indicated concentrations of VPA. Then 10 μL of 5 mg/mL MTT (purchased from Amresco Inc., OH, USA) in phosphate-buffered saline (PBS) was added to each well and the cells were incubated in the dark for 4 h at 37˚C. The cell culture medium was replaced with 150 μL of dimethyl sulfoxide, and the mixture was stirred for 10 min. The optical density (OD) values for samples were measured using a Pan-wavelength microplate reader at 570 nm. Each test included a blank containing a complete culture medium without cells. Triplicate wells were used for each sample, and the experiments were repeated at least three times to get means and standard deviations.
The cell viability was examined using the wound-healing assay. PC-3 cells in each group were seeded into a 6-well plate. When the cells reached 60%-80% confluence, a wound was built across the cell monolayer using a BioClean 1000 μL plastic pipette tip. The cells were rinsed with PBS three times and incubated with fresh medium containing different dosages of VPA for another 4 days or 2.0 mM VPA for another 2, 4, and 6 days. The cell proliferation and migration into the wound area were photographed under an inverted microscope (Leica, Wetzlar, Germany) with 100× magnification. The relative migration rate was calculated using the following formula: relative migration rate = (distance between the gap at 0 h - distance between the gap at each time point)/distance between the gap at 0 h × 100%.
Oil-Red O staining
The adherent PC-3 cells grown on a glass coverslip at the bottom of a 6-well plate were fixed in 4% paraformaldehyde for 15 min and then analyzed with an Oil-Red O stain kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the supplier’s instructions. After being rinsed in 60% isopropanol to remove unbound dye and counterstained with hematoxylin, the samples were taken photo of under an Olympus BX53 microscope (Olympus Corporation, Tokyo, Japan) equipped with a DP72 microscope digital camera and Image-Pro Plus 7.0 software. In order to quantify lipid accumulation, Oil-Red O was extracted with 100% isopropanol, and the light absorbance of the solution was measured at 520 nm.
Western blotting analysis
The cultured cells were collected and solubilized using protein lysis buffer. Lysates were cleared by centrifugation, and proteins were separated by gel electrophoresis. The membranes were blocked in PBS containing 0.1% Tween 20 and 5% (w/v) milk for 2 h at room temperature. The blots were first incubated with primary antibodies: anti-C/EBPα, anti-SREBP-1 (Abcam, Cambridge, UK), anti-Flag (Sigma-Aldrich Corp, St Louis, USA), anti-FASN, anti-acetyl CoA carboxylase 1 (ACC1), anti-B cell lymphoma 2 (Bcl-2), or anti-β-Actin (Proteintech, Wuhan, China), followed by incubation with the appropriate secondary antibody, that is, goat anti-rabbit IgG (H + L)-HRP or goat anti-mouse IgG (H + L)-HRP (RayBiotech, Beijing, China). The detection was performed using an enhanced chemiluminescence kit (Advansta, CA, USA).
DAPI staining assay
PC-3 cells (4 × 105 per well) were cultured in a 6-well plate, and nuclear morphology was tested using a DAPI staining assay. Following treatment with VPA (0, 1.0, 2.0, 5.0, and 10.0 mM) for 4 days, the cells were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.1% Triton X-100 for 2 min. Then they were stained with 10 μg/mL DAPI (purchased from Roche Corporation, Basel, Switzerland) at 37°C in the dark for 10 min. The nuclear morphology was viewed under ultraviolet light, and the images were captured using an inverted fluorescence microscope (Leica, Wetzlar, Germany). Apoptotic cells were identified according to characteristic changes, including nuclear condensation, fragmentation, and presence of apoptotic bodies .
Annexin V-FITC and PI double-staining assay
PC-3 cells (4 × 105 per well) were seeded in a 6-well plate overnight and treated with various concentrations of VPA for 4 days. In order to measure the extent of cell apoptosis, an Annexin V-fluorescein isothiocyanate (FITC) and PI double-staining apoptosis detection kit was used according to the manufacturer's instructions (Sungene Biotech, Tianjin, China). In brief, the cells were harvested and suspended in 1× binding buffer. Then, 100 μL of the cell suspension was incubated with 5 μL of Annexin V-FITC for 10 min, followed by incubation with 5 μL of PI solution for another 5 min. The labeled cells were then assessed by flow cytometry and analyzed using Cellquest 6.0 (BD Biosciences, NJ, USA) .
In brief, the PC-3 cells seeded in a 6-well, 12-well, or 96-well plate were transfected using Lipofectamine 3000 transfection reagent (Invitrogen, CA, USA) according to the manufacturer's protocol. The following plasmids were used: pcDNA3-C/EBPα (a gift from Dr. Hong Yin), pcDNA3.1-2xFlag-SREBP-1a, and pcDNA3.1-2xFlag-SREBP-1c (from Addgene, MA, USA). The siRNA against C/EBPα (in short, Si-C/EBPα) and the negative control siRNA (in short, Si-Control) were designed and synthesized by RiboBio Company (Guangzhou, China). The siRNA against C/EBPα used in the present study was as follows: sense: 5′-GGAGCUGACCAGUGACAAUdTdT-3′; antisense: 5′-AUUGUCACUGGUCAGCUCCdTdT-3′.
Quantitative real-time PCR
Total RNA was extracted from PC-3 cells after transfection with C/EBPα-expressing plasmid or Si-C/EBPα and/or treatment with VPA (2.0 mM) using an E.Z.N.A. HP Total RNA Extraction Kit (Omega Bio-Tek, GA, USA). Reverse transcription reactions were performed with 2.0 μg total RNA using a RevertAid First Strand cDNA Synthesis Kit and oligo (dT) primers (Fermentas Inc., Burlington, Ontario, Canada) according to the manufacturer’s protocol. The abundance of mRNA was detected using an SYBR Green real-time PCR kit according to the manufacturer’s instructions (Sangon Biotech, Shanghai, China). β-Actin reference gene was amplified in separate triplicate reactions for each sample and the average Ct value was calculated for the quantification analysis. The relative transcript quantities of SREBP-1a and SREBP-1c were measured using the comparative Ct (2−ΔΔCt) method. The measurement was performed in three independent experiments and each with three replicates. The sequences of the primer pairs were as follows: SREBP-1a: 5¢-CGGCGCTGCTGACCGACATC-3¢ and 5¢-CCCTGCCCCACTCCCAGCAT-3¢; SREBP-1c: 5¢-GCGCAGATCGCGGAGCCAT-3¢ and 5¢-CCCTGCCCCACTCCCAGCAT-3¢; and β-Actin: 5¢-CCAGAGATGGCCACGGCTGCT-3¢ and 5¢-TCCTTCTGCATCCTGTCGGCA-3¢.
The experiments were repeated at least three times, and data were analyzed using SPSS (version 17.0; SPSS, Inc., IL, USA). All data were expressed as the mean ± S.D. A statistical analysis between two groups was performed using the Student¢s t-test. One-way analysis of variance was used for the bar plots containing three or more groups. *P < 0.05, **P < 0.01, and ***P < 0.001 indicate significant differences, whereas the nonsignificant difference is denoted by NS.