Tgf βri

Tubastatin A was purchased from Selleckchem (Houston, TX, USA). Antibodies to p-STAT3 (Tyr-705), STAT3, p-Smad3 (Ser423/425), Smad3, p-EGFR (Tyr1068), p-NF-κB (Ser536), NF-κB, Histone H3, Acetyl Histone H3 (Lys9), Acetyl α-tubulin (Lys40) and HDAC6 were purchased from Cell Signaling Technologies (Danvers, MA, USA). Antibodies to collagen I (A2), E-cadherin, TGF-βRI, CD68, EGFR, VEGF, CD31 and GAPDH (glyceraldehyde 3-phosphate dehydrogenase) as well as HDAC6 siRNA were purchased from Santa Cruz Biotechnology (San Diego, CA, USA). Antibody to β-actin was purchased from TransGen Biotech (Beijing, China). TNF-α, IL-1β, TGF-β1, MCP-1, IL-6 and TGF-β1 enzyme-linked immunosorbent assay (ELISA) kits were from R&D Systems (Minneapolis, MN, USA). α-SMA, DMSO and other chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA).

For immunocytochemistry, serum-starved MEFs were grown on glass slides, treated with TGF-β1, fixed with cold 4% paraformaldehyde (PFA) for 15 min, and then incubated in NH₄Cl/PBS (50 mM) for 10 minutes. Cells were permeabilized with 0.25% Triton X-100, blocked in 3% BSA for 30 minutes at room temperature, and incubated with primary antibodies at 4°C overnight. Cells were immunolabeled with the following antibodies: Smad2 (Santa Cruz), Smad3 (Cell Signaling), α-SMA (Sigma), TGF-β RI (Santa Cruz), TGF-β RII (Santa Cruz), and EEA1 (BD Biosciences). FITC- or rhodamine-conjugated secondary antibodies were used, and nuclei were counterstained with Hoechst 33342 (Invitrogen). Mounted cells were analyzed by confocal laser scanning microscopy using a Zeiss LSM Meta 510. For immunohistochemistry, skin samples were fixed in 4% PFA, embedded in paraffin, and sectioned. Sections were heated in DAKO Target Retrieval Solution (pH 6.0) for antigen retrieval, blocked in 5% BSA for 1 hour at room temperature, and incubated with Smad2 (Santa Cruz) primary antibody at 4°C overnight. Samples were incubated with HRP-conjugated secondary antibody and exposed to DAB for colorimetric detection. Nuclei were counterstained with hematoxylin. Mounted samples were analyzed by digital fluorescent microscopy.

DEN was purchased from Sigma (St. Louis, MO, USA). Antibodies for transforming growth factor-beta receptor (TGF-βR) I, TGF-βRII, phospho-EGFR, and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies for phospho-Smad2, phospho-Smad3, Smad2/3, phospho-STAT3, STAT3, phospho-Akt, Akt, phospho-Jak2, Jak2, phospho-Src, and Src were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies for proliferating cell nuclear antigen (PCNA), antigen Ki-67 (Ki67), glutathione S-transferase pi (GST-pi) and α-SMA were purchased from Abcam (Cambridge, UK).

The protein extracts from the frozen heart tissues and the si-transfected cells were separated by a SDS PAGE (12%) and transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA, USA), followed by the blocking of the non-specific protein with 5% dried milk for 1 h. The blocked membranes were incubated overnight at 4 °C with the primary antibody against IRF3 (cell signaling), TGFβ (Sigma, Burlington, MA, USA), TGFβ-RI, TGFβ-RII, p53, p16, Bax and BCL, and GAPDH (all Santa Cruz). The detection was performed using the suitable horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature in 5% dried milk. The plots were quantified by densitometry and normalized against GAPDH.

The following compounds were used in this study: DATS (purity > 97%; Shenzhen Minn Bolin Biotechnology Co., Ltd., Shenzhen, China), iodoacetamide (IAM; Nanjing Dingguo Changsheng Biotechnology Co., Ltd., Nanjing, China), and mitomycin (Sigma, St Louis, MO, USA). They were dissolved in dimethylsulfoxide (DMSO) for experiments. DMSO at a concentration of 0.02% (w/v) was set as a vehicle control throughout the studies. Analytical grade 30% hydrogen peroxide (H₂O₂; Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) was diluted with deionized water to indicated concentrations for experiments. The following primary antibodies were used in this study: α-SMA, α1(I) procollagen, and fibronectin (Epitomics, San Francisco, CA, USA); TGF-βRI, TGF-βRII, PDGF-βR, EGF-R, and Bcl-2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); cyclin A, cyclin B1, CDK1, CDK2, Bax, pro-caspase-9, cleaved-caspase-9, pro-caspase-8, cleaved-caspase-8, pro-caspase-7, cleaved-caspase-7, pro-caspase-3, cleaved-caspase-3, full-length PARP-1, cleaved-PARP-1, and β-Actin (Cell Signaling Technology, Danvers, MA, USA).

Cell-culture reagents were from Gibco (Grand Island, NY, USA). Lipofectamine and TRIzol were from Invitrogen (Carlsbad, CA, USA). G418 was from Merck (Darmstadt, Germany). Monoclonal antibody to IGFBP-rP1 and recombinant human IGFBP-rP1 protein were from RD (Minneapolis, MN, USA). Mouse monoclonal antibodies to E-cadherin, N-cadherin, TGF-β R I, TGF-β R II, β-catenin, actin, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Polyclonal antibodies to vimentin, MMP9, Snail, Smad2, P-Smad2, Smad3, and P-Smad3 were from Cell Signaling (Danvers, MA, USA). Recombinant human TGF-β1 was from Peprotech (Rocky Hill, NJ, USA). SB431542 (TGF-β/ALK5/Smad2 inhibitor) was obtained from Sigma.