Ag490

Esophageal squamous cell carcinoma (ESCC) cell lines EC1, EC9706, KYSE450 and TE13 were provided by Department of Pathophysiology, School of Basic Medicine, Zhengzhou University. All ESCC cell lines were cultured in Dulbecco’s high glucose modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 μg/ml of penicillin, and 100 units/ml of streptomycin at 37 °C with 5% CO₂.
The ESCC tumors used for this study were collected from patients enrolled into the First Affiliated Hospital of Zhengzhou University (Zhengzhou, China) with consensus, and approved by the Ethics Committee of Zhengzhou University. None of these patients had received preoperative chemotherapy or preoperative radiation therapy. The fresh tumor specimens were collected at the time of surgical resection and prepared for implantation in immunodeficient mice. All specimens were examined by two pathologists to confirm the malignant tissues. All the tissues were inoculated into the mice within 2 h after the operations.
Curcumin, Z-VAD-FMK and AG490 were purchased from Selleck Chemicals (Houston, TX, USA). Annexin V-FITC Apoptosis Detection Kit was purchased from Beyotime Biotechnology (Shanghai, China).

Anti-GRP78, PERK, phosphor-PERK, JAK2, and STAT3 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). We purchased thapsigargin from Sigma (USA) and GSK2606414 and AG490 from Selleck Chemicals (USA). TGF-β1 and FN ELISA kits were bought from Boster (Wuhan, China), a collagen I ELISA kit was from BlueGene Biotech. (Shanghai, China), and a cell counting kit (CCK-8) was bought from Dojindo (Kumamoto, Japan). Rat renal proximal tubule epithelial cells (NRK-52E) were obtained from the American Type Culture Collection.

Human HCC cell lines, SMMC-7721 and BEL-7402 and immortalized normal liver cell line LO2 were obtained from Shanghai Institute of Cell Biology, Chinese Academy of Sciences. MHCC-97L was a gift from Fudan University (Dr. ZY Tang) of Shanghai. BEL-7402 and MHCC-97L cells are hepatitis B surface antigen (HBsAg) positive. SMMC-7721, BEL-7402, MHCC-97L and LO2 were maintained in Dulbecco's modified Eagle's medium with high glucose (Gibco-BRL, Carlsbad) supplemented with 10% fetal bovine serum. Y27632, MG132, AG490 and BAY11-7082 were purchased from Selleckchem (Houston). Cisplatin and doxorubicin were purchased from Pharmachemie BV (Haarlem). The plasmids for STAT3 and NFκB luciferase reporter assays were constructed by cloning STAT3 and NFκB binding elements into pGL3(Promega, Wisconsin) using NheI and BgIII.

Anti-KSHV LANA rat monoclonal antibody (MAb) was purchased from Advanced Biotechnologies Inc.(Columbia, MD, USA) [81 (link)]. Anti-phospho-STAT3 (Y705) rabbit MAb, anti-STAT3 mouse polyclonal antibody (PAb), and anti-Flag M2 rabbit MAb were obtained from Cell Signaling Technologies (Beverly, MA, USA). Anti-SH3BGR mouse MAb, anti-GAPDH mouse MAb, anti-α-Tubulin mouse MAb, and horseradish peroxidase (HRP)-conjugated goat anti-mouse or anti-rabbit IgG were all purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-His mouse MAb, anti-mouse immunoglobulin G (IgG) and anti-GST mouse MAb were from Beyotime Institute of Biotechnology (Nantong, Jiangsu, China). Western blotting analysis was performed as described previously [84 (link), 85 (link)]. AG490, a JAK2 inhibitor, was purchased from Selleck Chemicals (Shanghai, China).

Different inhibitors of specific signal transduction pathways, including Vandetanib (ZD6474), U0126, PP1, AG490 and S3I-201, were purchased from Selleck. Phosphor-Ret(Tyr⁹⁰⁵), Ret, phospho-STAT3 (Tyr⁷⁰⁵), Phospho-STAT3(Ser⁷²⁷), STAT3, phospho-ERK1/2(Thr²⁰²/Tyr²⁰⁴), ERK_1/2, glyceraledehyde-3-phosphatedehydrogenase (GAPDH), and anti-Flag antibodies were purchased from Cell Signaling Technology. STAT3 recombinant protein was purchased from Abnova.

This research was in agreement with the Declaration of Helsinki. The cell line used in this study was mouse L929, which was purchased from the cell bank of the Shanghai Biology Institute (Shanghai, China). Cells were cultured in DMEM medium (Trueline, USA) and grown in a 5% CO₂ condition at 37°C. Then, cells were cultured by the mouse recombinant protein CXCL16 (Abcam, UK) and the STAT3 inhibitor AG490 (Selleck, USA) for 48 h with different concentrations, including 50, 100, and 200 ng/ml, respectively.

Ten 4-week-old male C57BL/6J mice were purchased from Shanghai JSJ Laboratory Animal Co., Ltd., which were randomly divided into two groups. Vehicle group: mice received daily intraperitoneal injections of DPBS (Corning, 21-031-CMR) for 5 weeks. AG490 group: mice received daily intraperitoneal injections of AG490 (5 mg/kg; Selleck, S1143) for 5 weeks. The femurs of the 9-week-old mice were then used for micro-CT or histological analysis.