P akt

Human prostate stromal cell line WPMY-1 was kindly provided by Stem Cell Bank, Chinese Academy of Sciences (Shanghai, China), and cultured in DMEM (Hyclone, USA) containing 5% fetal bovine serum (Gibco, USA) with 1% penicillin G sodium/streptomycin sulfate at 37 °C with 5% CO₂. MK2206, which is a phosphorylated Akt (p-Akt) inhibitor (Beyotime Biotechnology, Shanghai, China). For IL-6Rα (interleukin-6 receptor-α) knockdown, siRNAs and the corresponding controls were purchased from the GenePharma (Shanghai, China) and transfected into cells with Lipofectamine 2000 (Invitrogen, CA, USA). The target sequences are shown in Additional file 1: Table S2.

Tetrahydrocurcumin was purchased from Yuanye Biotechnology Co. Ltd (Shanghai,China). E-cadherin, ZEB, Snail, Twist, HIF-1α, p-mTOR, mTOR, p-ERK, ERK, GAPDH and β-actin antibodies were purchased from Santa cruz Biotechnology (Santa cruz, CA, USA). Vimentin, N-cadherin, VEGF, MMP-2, MMP-9, LaminA/C, Atg5, Atg7 and Beclin-1 antibodies were obtained from Boster Biological Technology Co. Ltd (Wuhan, China). LY294002, SB203580, Rapamycin, β-catenin, p-Akt, Akt, p-JNK, JNK, p-p38, p38, IκB-α, p65, Hsp90 and GSK-3β antibodies were obtained from Beyotime Institute of Biotechnology (Haimen, China). Antibody against LC3B was purchased from Abcam (Cambridge, UK). All other reagents were from common commercial sources.

Insulin and antibodies against IRS-1, pIRS-1 (Ser307), PTEN, Akt, pAkt (Ser473), GSK3β and pGSK3β (Ser9) were purchased from Beyotime, China. GAPDH antibody was from Abmart, Shanghai. HCV core monoclonal antibody was obtained from Abcam. Horseradish peroxidase (HRP)-conjugated secondary antibodies were from Santa Cruz Biotechnology.

Total proteins were extracted from PAs of animal models and PASMCs with different treatments. RIPA (P0013B, Beyotime) contained PMSF (ST506, Beyotime) were added to lyse tissue or cells, and then centrifuged at 4°C, 14,000 rpm for 10 min. The supernatant was collected for Western blot experiments. 50 μg samples were isolated with a 10% SDS polyacrylamide gel and then transferred to PVDF membranes. After blocking with 5% skim milk for 2 h, the membranes were incubated with primary antibodies against Tex261 (1:500, Novus), Hif-1a, PCNA, Ndgr1, p-Akt, Akt (1:500, Beyotime) and Sec23a (1:5000, Abcam) at 4 °C overnight. After secondary antibodies were incubated for 2 h, the blots were reacted with enhanced chemiluminescence (ECL) reagent (Thermo, United States) for 5 min. Exposure detection was performed under a Biomolecular Imager. The relative optical density was analyzed, and β-actin was used as the control.

Total cellular proteins were extracted from duck myoblasts with RIPA lysis buffer (Beyotime Biotech, China). Protein samples were resolved by 10% SDS/PAGE and electroblotted on PVDF membranes (Beyotime Biotech, China). The membranes were incubated in block buffer (Beyotime Biotech, China) at 37°C for 2 h and then incubated with the primary antibody at 4°C for 12 h. After that, the membranes were incubated with a secondary antibody at 37°C for 2 h, and subsequently detected by using the ECL kit (Beyotime Biotech, China) and a Gel Imaging System (Bio-Rad, U.S.A.). Primary antibodies against the following proteins were purchased from Biosynthesis Biotechnology and used at 1:1000 dilutions: p38α, p-p38α, MRF4, Akt, p-Akt, while GAPDH (diluted 1:1000) was purchased from Beyotime Biotech. The secondary antibodies (HRP–conjugated goat anti-rabbit IgG or goat anti-mouse IgG) were purchased from Biosynthesis Biotechnology as well. ImageJ software (National Institutes of Health, U.S.A.) was used for densitometry analysis. The values below each Western blot image represent the relative abundance of the target protein compared with GAPDH.

Proteins were abstracted from liver tissue using a lysis solution (Beyotime, Shanghai, China) with 0.5 mM PMSF and phosphatase inhibitors. Protein concentrations were determined to calculate the loading amount. SDS-PAGE was performed on proteins using a 10% acrylamide gel. The specific operation process of protein immunoblotting also referred to a previous study [23 (link)]. Antibodies against t-Akt, p-Akt, SREBP-1c, and β-actin were from Beyotime Biotech, Shanghai, China. The secondary antibodies were from Proteintech Group, Inc., Wuhan, China.

AT-II (purity ≥ 98%) was purchased from Must Bio-technology Co., Ltd. (Chengdu, China). Dimethyl sulfoxide (DMSO) was purchased from Sigma (St. Louis, MO, USA). CCK-8 Kit and human Bcl-2, Bax, Akt, p-Akt, ERK, p-ERK and β-actin polyclonal antibodies were purchased from Beyotime (Shanghai, China). Annexin V-FITC Apoptosis Detection Kit was purchased from KeyGEN BioTECH (Nanjing, China).