Anti-C/EBP-α (cat. no. sc-61-G) was obtained from Santa Cruz Biotechnology, Inc. (Dallas, Texas, USA), horseradish peroxidase (HRP)-conjugated goat anti-mouse immunoglobulin (Ig)G (cat. no. SA0001-1) and goat anti-rabbit IgG (cat. no. SA0001-1) were obtained from ProteinTech Group, Inc. (Chicago, IL, USA). Anti-β-actin (A5441) was purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Antibodies against Caspase-3 (cat. no. #9662, which recognizes the procaspase and cleaved Caspase forms), cleaved Caspase-3 (cat. no. #9661, which detects only the cleaved form), Caspase-8 (cat. no. #4790, which recognizes the procaspase and cleaved Caspase forms), Caspase-9 (cat. no. #9508, which recognizes the procaspase and cleaved Caspase forms), and acetylated lysine (cat. no. #9441) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Anti-Caspase-12 (cat. no. 3282-100, which recognizes the procaspase and cleaved Caspase forms) was purchased from BioVision, Inc. (Milpitas, CA, USA). The Cell Counting Kit-8 (CCK-8) assay kit was purchased from Dojindo Molecular Technologies, Inc. (Kumamoto, Japan). NE-PER nuclear and cytoplasmic extraction reagents and the Pierce Co-Immunoprecipitation (co-IP) kit were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). TRIzol reagent was obtained from Sangon Biotech Co., Ltd. (Shanghai, China). The PrimeScript RT Reagent kit (Perfect Real Time) and SYBR Premix Ex Taq kit (Perfect Real Time) were obtained from Takara Biotechnology Co., Ltd. (Dalian, China). TSA and nicotinamide were purchased from Sigma-Aldrich (Merck KGaA). SAHA was obtained from Cayman Chemical Company (Ann Arbor, MI, USA). Epigallocatechin gallate (EGCG) was obtained from YuanYe Biotechnology Co., Ltd. (Shanghai, China).

The immortalized rat liver stellate cell line HSC-T6 was established by Dr. Scott L. Friedman and generously provided by his student, Professor Lie-Ming Xu (Shanghai University of Traditional Chinese Medicine, Shanghai, China). The rat hepatocyte cell line BRL-3A was purchased from the American Type Culture Collection (Manassas, VA, USA). The two cell lines were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (both from Gibco; Thermo Fisher Scientific, Inc.) and standard antibiotics in a 95% air and 5% CO₂ humidified atmosphere at 37°C.

The plasmid PDC316 was constructed in the lab previously (4). Adenovirus construction, amplification, purification and titer detection were performed as described previously (4).

HSC-T6 and BRL-3A cells were seeded onto 96-well plates at a density of 1×10⁵ cells/well. Cells were treated with varying concentrations of TSA, SAHA and nicotinamide for 48 h at 37°C (Fig. 1). Cell viability was measured using the CCK-8 kit. Control cells were treated with 10% dimethyl sulfoxide for 48 h. Following the addition of the test compounds, 10 µl CCK-8 was added to each well, incubated for 1–3 h at 37°C, and optical density was read at a wavelength of 450 nm in a VMax kinetic microplate reader (Molecular Devices, LLC, Sunnyvale, CA, USA).

Following treatment with TSA for the indicated times (0, 1, 2, 4, 8, 12, 24, 36 and 48 h) at concentration of 0.1 µmol/l, total RNA from HSC-T6 cells was extracted using TRIzol reagent, according to the manufacturer's protocol. Briefly, total RNA (1 µg) was reverse-transcribed using a PrimeScript RT kit (Takara Biotechnology Co., Ltd.) with oligodT primer and six random primers, to obtain the cDNA. The cDNA was amplified by PCR. PCR was performed in a Rotor Gene thermal cycler (RG-3000; Qiagen GmbH, Hilden, Germany) under conditions optimized for efficient amplification of the respective genes and the reference gene. Amplification was performed as follows: Following the initial activation step at 95°C for 5 min, 40 cycles of denaturation at 95°C for 5 sec and annealing at 60°C for 30 sec were performed. qPCR (11) experiments were conducted in three biological replicates. The following primer sequences were used: Rat C/EBP-α, 5′CGG TGG ATA AGA ACA GCA ACGA3′ (sense) and 5′GCG GTC ATT GTC ACT GGT CAAC3′ (antisense); and rat β-actin, 5′AGG ATG CAG AAG GAG ATT ACT GC3′ (sense) and 5′AAA ACG CAG CTC AGT AAC AGT GC 3′ (antisense).

Following treatment at 37°C with 0.1 µmol/l TSA for the indicated times (0, 1, 2, 4, 8, 12, 24, 36 and 48 h), or treatment with TSA and/or Ad-C/EBP-α with a range of dosages, HSC-T6 cells were collected, washed in cold PBS, and subsequently lysed in ice-cold radioimmunoprecipitation assay buffer [10 µM Tris-HCl (pH 8.0), 100 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, and 10 µl/ml protease inhibitor cocktail] on ice for 45 min. Lysates were cleared by centrifugation at 13,000 × g for 30 min at 4°C, and total protein concentration was determined using a Bicinchoninic Acid Protein Assay kit. Extracted cellular proteins (10 µg) from each sample were denatured in SDS containing sample buffer, applied to a 10–12% SDS-PAGE gel and transferred to nitrocellulose membranes. Following blocking in 5% non-fat milk for 1 h at room temperature, the membranes were incubated overnight at 4°C with primary antibodies against anti-C/EBP-α (1:200), anti-Caspase-3 (1:1,000), anti-Caspase-8 (1:1,000), anti-Caspase-9 (1:1,000) or anti-Caspase-12 (1:1,000), respectively. β-actin (1:1,000) was used as a loading control. Membranes were subsequently incubated for 1 h at 4°C with the appropriate HRP-conjugated secondary antibody. Protein bands were visualized using an Enhanced Chemiluminescence Assay kit (Thermo Fisher Scientific, Inc.). ImageJ 1.50e (National Institutes of Health, Bethesda, MD, USA) was used for densitometric analysis.

HSC-T6 cells (~1×10⁶) were harvested following treatment with 0.1 µmol/l TSA for 12 h at 37°C. Cells were washed in cold PBS, and nuclear and cytoplasmic proteins were extracted using NE-PER nuclear and cytoplasmic extraction reagents, according to the manufacturer's protocol. Proteins were stored at −80°C until ready for use in subsequent experiments.

Following treatment with TSA at a concentration of 0.1 µmol/l for 12 h at 37°C, co-IP analysis of C/EBP-α and the acetylated lysine antibody was performed in HSC-T6 cells using a co-IP kit, according to the manufacturer's protocol. C/EBP-α and the acetylated lysine antibody were coupled to AminoLink Plus Coupling Resin. HSC-T6 cell lysate was prepared with IP lysis/wash buffer on ice for 5 min. Purified HSC-T6 cell lysate was incubated with C/EBP-α, acetylated lysine antibody-specific MAb-conjugated agarose resin overnight at 4°C. Following elution, proteins samples were prepared for western blot analysis using antibodies against C/EBP-α or acetylated lysine as aforementioned. A sample of the whole input lysate served as a positive control, and normal mouse IgG (Beyotime Institute of Biotechnology, Haimen, China) was used as negative control.

All experiments were repeated three independent times. Statistical analysis was performed using SPSS software, version 11.0 (SPSS, Inc., Chicago, IL, USA). Data were expressed as the mean ± standard deviation. Comparisons were performed using Student's t-test. For multiple comparisons, repeated measures analysis of variance and Dunnett's test were used. P<0.05 was considered to indicate a statistically significant difference.

HSC-T6 and BRL-3A cells exhibited differential responses to the three HDACIs. HSC-T6 cells were more sensitive to TSA compared with BRL-3A cells. Proliferation was inhibited by 0.05 µmol/l and 0.2 µmol/l TSA. The rate of proliferation inhibition was 67.54% in HSC-T6 cells and 22.21% in BRL-3A cells (P<0.01). However, compared with treatment with TSA, BRL-3A cells were more sensitive to SAHA and nicotinamide (Fig. 1). The ideal medicine to treat liver fibrosis is one that is able to inhibit HSCs while exerting a small effect on hepatocytes. Therefore, TSA was selected for the subsequent experiments in HSC-T6 cells.

Varying doses of TSA alone or in combination with 100 multiplicity of infection (MOI) Ad-C/EBP-α were used to detect the expression of apoptotic markers of Caspase-3, −8, −9 and −12 levels in HSC-T6 cells (Fig. 2). It was observed that TSA alone and in combination with 100 MOI Ad-C/EBP-induced apoptosis in HSC-T6 cells. Treatment with TSA induced apoptosis through Caspase-9 and Caspase-12, whereas Ad-C/EBP-α induced apoptosis primarily through the Caspase-9 pathway. However, activated Caspase-8 was not detected. These results suggested that TSA may enhance C/EBP-α-induced apoptosis of HSCs.

In order to further investigate the effect of TSA on C/EBP-α expression in HSC-T6 cells, they were first treated with TSA at concentration of 0.1 µmol/l, and subsequently harvested for RT-qPCR and western blot analyses at 0, 1, 2, 4, 8, 12, 24, 36 and 48 h. RT-qPCR analysis demonstrated that C/EBP-α mRNA expression did not alter significantly, however it was observed that C/EBP-α protein expression increased and reached a peak between 4 and 12 h, prior to decreasing gradually, indicating that C/EBP-α may be processed post-transcriptionally (Fig. 3).

Following treatment with TSA for 12 h at a concentration of 0.1 µmol/l, cells were harvested to examine the nuclear and cytoplasmic proteins. C/EBP-α protein expression was decreased in the cytoplasm following treatment with EGCG, which served as the negative control (Fig. 4). However, it was observed that C/EBP-α protein expression was increased in the nuclear fraction following treatment with TSA, verifying that C/EBP-α predominantly functions in the nucleus.

Following treatment with TSA for 12 h at concentration of 0.1 µmol/l, cells were harvested to examine the lysine acetylation of C/EBP-α by co-IP analysis. As presented in Fig. 5, immunoprecipitation of cell lysates with the anti-acetylated lysine antibody coprecipitated with the C/EBP-α antibody, and the C/EBP-α antibody additionally coprecipitated with the anti-acetylated lysine antibody. Western blot analysis was performed to detect the expected bands; compared with untreated cells, lysine acetylation increased following treatment with TSA.