Monoclonal rabbit antibodies against human total p65 (cat. no. 8242), phospho (p-)p65 (cat. no. 3031), as well as monoclonal mouse antibodies against acetylated-Lysine (cat. no. 9681) and human β-actin (cat. no. 3700) were from Cell Signaling Technology (Danvers, MA, USA). Mouse monoclonal antibodies against human PCAF (cat. no. sc-13124) and Hemagglutinin (HA)-tag (cat. no. sc-7392) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). For western blot analysis, horseradish peroxidase-conjugated anti-mouse immunoglobulin G (IgG) antibody (cat. no. 7076) and anti-rabbit IgG antibody (cat. no. 7074) were purchased from Cell Signaling Technology. Enhanced chemiluminescence (ECL) Western Blotting Substrate, co-immunoprecipitation (co-IP) assay buffer, radioimmunoprecipitation assay (RIPA) lysis buffer, a bicinchoninic acid (BCA) protein assay kit and protein G-Sepharose beads were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Polyvinylidene difluoride (PVDF) membranes were obtained from Roche (Basel, Switzerland). TRIzol reagent was purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). Moloney Murine Leukemia Virus reverse transcrip-tase and oligo (dT) 15 primer were purchased from Promega (Madison, WI, USA). TaqMan^® Fast Advanced Master Mix was from Applied Biosystems (Thermo Fisher Scientific). The plasmids pGCsi-U6/neo/green fluorescence protein (GFP) were obtained from Shanghai Genkan Biotechnology Co., Ltd (Shanghai, China). The pcDNA3.1 vector was purchased from Invitrogen Life Technologies. The incision enzymes HindIII and XhoI as well as T4 DNA ligase were purchased from Takara Bio Inc. (Tokyo, Japan). The Escherichia coli strain DH5α was purchased from Molecular Cloning Laboratories (San Francisco, CA, USA). QIAprep spin miniprep kit was obtained from Qiagen (Hilden, Germany). PDTC was purchased from Abcam (Cambridge, UK).

The human AGS cells were obtained from the American Type Culture Collection (Manassas, VA, USA). Cells was cultured in the medium of RPMI-1640 (Gibco-BRL; Invitrogen Life Technologies) supplemented with 10% fetal bovine serum (FBS; Gibco-BRL) and antibiotics (50 U/ml penicillin and 100 μg/ml streptomycin) at 37°C in a 5%-CO₂ incubator. AGS cells were incubated with Helicobacter pylori CagA protein at 37°C for different time points. For certain studies, the cells were incubated with 10 μM PDTC at 30 min before treatment with Helicobacter pylori CagA protein.

The open reading frame of human PCAF mRNA (National Center of Biotechnology Information reference sequence, NM_003884.4) containing a HA tag was amplified by polymerase chain reaction (PCR) from cDNA of human AGS cells. The PCR products and pcDNA3.1 vector were further digested with the two restriction enzymes of HindIII and XhoI, and then ligated with each other by using T4 DNA ligase. The recombinant plasmids were amplified in the Escherichia coli strain DH5α at 37°C for 16 h. Subsequently, the plasmids were extracted by QIAprep spin miniprep kit according to the manufacturer's instructions. Finally, the constructed plasmids (pcDNA3.1/PCAF-HA) were sequenced to confirm the nucleotide sequence by GenScript (Nanjing, China).

Various shRNA sequences targeting human PCAF mRNA (NM_003884.4) were designed to silence the PCAF gene in human AGS cells. The various DNA segments for the expression of PCAF shRNA were synthesized and inserted into the shRNA pGCsi-U6/neo/GFP plasmids by Shanghai Genkan Biotechnology Co., Ltd. The most effective shRNA expression plasmid to silence human PCAF gene was selected to be used in subsequent experiments. The PCAF shRNA sequence was as follows: AAG ATG GCC GTG TTA TTG GTG and the Scrambled shRNA sequence was as follows: AAT GAC GGG CTT GTT ATG GGT.

Plasmids were transfected into AGS cells by using Lipofectamine 2000 according to the manufacturer's instructions. For transfection, 4 μg plasmid was mixed with 250 μl serum-free RPMI-1640. At the same time, 10 μl Lipofectamine 2000 was mixed with 250 μl serum-free RPMI-1640. The plasmids and Lipofectamine 2000 were further incubated with each other for 20 min at room temperature. Finally, the 500-μl mixture was added into each well in a 6-well plate containing 4×10⁵ AGS cells. The medium was replaced with serum containing RPMI-1640 at 5 h after transfection, and the cells were incubated sequentially.

Helicobacter pylori CagA-His-tag was constructed in the pET21a plasmid from Novagen (Madison, WI, USA). The plasmid was then transformed into BL21(DE3) Singles™ Competent Cells (Novagen) for protein expression and purification according to the manufacturer's instructions. The protein was extracted by B-PER^® bacterial protein extraction reagent (Thermo Fisher Scientific) from 50 ml bacteria according to the manufacturer's instructions. Subsequently, HisPur™ Cobalt Spin Columns (Thermo Fisher Scientific) were used for His-tag purification of the Helicobacter pylori CagA protein according to the manufacturer's instructions.

AGS cells were lysed in co-IP assay buffer at 4°C for 30 min. Cell lysates were then centrifuged at 15,000 ×g for 20 min at 4°C to remove any insoluble material. 300 μg cell lysate was incubated with 40 μl protein G-Sepharose beads in co-IP assay buffer at 4°C for 2.5 h under constant agitation and then centrifuged at 1,000 × g for 2 min at 4°C. The recovered supernatant was then incubated with the antibody against human total p65 (1.5 μg for each sample) at 4°C overnight under constant agitation. The sample was then incubated with 40 μl protein G-Sepharose beads at 4°C for 2.5 h under constant agitation. Protein G-protein complex was precipitated by centrifugation and finally re-suspended in 40 μl SDS lysis buffer (Beyotime Institute of Biotechnology, Haimen, China). The samples were then analyzed by SDS-PAGE followed by staining with specific antibodies against total p65 (1:1,000), acetylated-Lysine (1:1,000), PCAF (1:200) and HA (1:500), respectively. At the same time, 40-μg aliquots of whole-cell extract from the AGS cells were used to detect the protein expression as an input control.

Cells were collected at the indicated time-points and RNA was extracted using TRIzol reagent. A total of 1 μg RNA was used for the first-strand cDNA synthesis using MMLV and oligo (dT) 15 primer according to the manufacturer's instructions. The cDNA was amplified by using TaqMan^® Fast Advanced Master Mix to detect the expression of human PCAF gene using primers purchased from GenScript (forward 5′-TAC CTC GGT ACG AAA CCACA-3′ and reverse 5′-TCC TGT CTT GCT TGT TCCAG-3′; Fam/Tamra-labeled probe, 5′-CGA GCG AAG CAA TGT TCT CCCA-3′). The human β-actin gene was used as an internal control using primers purchased from GenScript (forward 5′-TGG ACT TCG AGC AAG AGATG-3′ and reverse 5′-GAA GGA AGG CTG GAA GAGTG-3′; Fam/Tamra-labeled probe, 5′-CGG CTG CTT CCA GCT CCTCC-3′). The 7500 Real-time PCR system (Applied Biosystems) was used to perform qPCR. The reaction program included an initial step for denaturation at 95°C for 10 min, and 40 cycles of denaturation at 95°C for 15 sec and annealing at 60°C for 60 sec. Each sample was assayed in triplicate. The relative levels of the gene expression were obtained using the 2^−ΔΔCt method as described previously (38).

Cells were lysed in RIPA lysis buffer at 4°C for 30 min. The cellular lysates were then centrifuged at 15,000 x g for 20 min at 4°C to remove any insoluble material. The concentration of the protein was determined by a BCA protein assay kit according to the manufacturer's instructions. The protein (40 μg/well) was subjected to 10–12% SDS-PAGE and transferred onto PVDF membranes using the Mini-Protean System (Bio-Rad Laboratories, Inc., Hercules, CA USA). The PVDF membranes were incubated in blocking buffer [5% skimmed milk in Tris-buffered saline containing Tween 20 (TBS-T)] at room temperature for 1 h and then incubated with the specific antibodies (anti-total p65, 1:1,000; anti-p-p65, 1:1,000; anti-acetylated-Lysine, 1:1,000; anti-PCAF, 1:200; anti-HA; 1:500) at 4°C overnight. β-actin expression in each sample was used as an internal standard (anti-β-actin, 1:1,000). After five washes with TBS-T, the PVDF membranes were further incubated with horseradish peroxidase-conjugated anti-mouse IgG (1:2,000) or anti-rabbit IgG (1:2,000) at 37°C for 1 h. The bands were visualized using X-ray film (Life Technologies, Carlsbad, CA, USA) and the ECL detection system after washing the PVDF membranes five times. Finally, the density of the radiographic bands on the PVDF membranes was analyzed using the Quantity One software v44 (Bio-Rad Laboratories, Inc.).

The levels of TNF-α and IL-6 in the cell media were determined using commercial human TNF-α and IL-6 ELISA kits (cat. no. 555212 and 550799) from BD Biosciences (Franklin Lakes, NJ, USA). ELISA was performed according to the manufacturer's instructions.

All statistical analyses were performed using SPSS 12 software (SPSS, Inc., Chicago, IL, USA). Values are expressed as the mean ± standard deviation. The statistical significance of differences between groups was evaluated by one-way analysis of variance with simultaneous multiple comparisons between groups by the Bonferroni method. P<0.05 was considered to indicate statistically significant differences.

Human AGS cells were cultured with purified Helicobacter pylori CagA and the production of TNF-α and IL-6 was subsequently observed. The results showed that CagA enhanced the production of TNF-α and IL-6 in human AGS cells in vitro in a dose- and time-dependent manner (Fig. 1). Furthermore, CagA was able to induce the activation of NF-κB in human AGS cells in vitro (Fig. 2A). Of note, inhibition of NF-κB significantly reduced the production of TNF-α and IL-6 in AGS cells induced by CagA (Fig. 2B-E). These findings indicated that CagA promoted the production of TNF-α and IL-6 by human AGS cells in vitro via activation of NF-κB.

Since Helicobacter pylori CagA was found to have the ability to stimulate the activation of NF-κB and the production of TNF-α and IL-6 in human AGS cells (Figs. 1 and 2), the effects of CagA stimulation on the acetylation of p65 were further assessed in AGS cells. The level of p65 acetylation was found to be significantly enhanced in human AGS cells exposed to CagA in vitro (Fig. 3), suggesting that p65 acetylation may have an important role in promoting p65 activation in human AGS cells induced by CagA.

The expression of PCAF at the mRNA and protein level was detected in human AGS cells induced by CagA. It was found that incubation with CagA significantly increased the mRNA and protein expression levels of PCAF in human AGS cells (Fig. 4A and B). Furthermore, the interaction of PCAF with p65 was assessed at the protein level by IP. The results showed that the molecular interaction of PCAF with p65 at the protein level was markedly enhanced in human AGS cells upon stimulation with CagA (Fig. 4B). These findings indicated that stimulation with CagA induced the expression of PCAF and further enhanced the molecular interaction of PCAF with p65 at the protein level in human AGS cells, suggesting a potential ability of PCAF to acetylate p65.

To further determine the role of PCAF expression in p65 acetylation and phosphorylation, AGS cells were treated with PCAF shRNA expression vector for 36 h followed by CagA stimulation for 1.5 h. A Co-IP assay showed that PCAF knockdown decreased CagA-induced p65 acetylation and phosphorylation in AGS cells (Fig. 5A). Conversely, overexpression of PCAF triggered p65 acetylation and phosphorylation in AGS cells (Fig. 5B). These results suggested that PCAF is necessary for p65 acetylation and phosphorylation in CagA-induced AGS cells.

Since PCAF was shown to be required for p65 acetylation in AGS cells stimulated by CagA (Fig. 5), the present study further explored the role of PCAF-mediated p65 acetylation in the production of TNF-α and IL-6 in AGS cells induced by CagA. The results showed that suppression of PCAF markedly reduced the production of TNF-α and IL-6 in AGS cells stimulated by CagA (Fig. 6A-D). Conversely, AGS cells overexpressing PCAF exhibited enhanced production of TNF-α and IL-6 (Fig. 6E-H). These results indicated that PCAF-mediated p65 acetylation is involved in the production of TNF-α and IL-6 in AGS cells stimulated with CagA.