Sistat3

WI38 cells were purchased from American Type Culture Collection. Cells were cultured in DMEM supplemented with 10% FBS (R&D Systems) and 1% Penicillin-Streptomycin (ThermoFisher Scientific). These cells were screened for mycoplasma every four months using the ATCC Universal Mycoplasma Detection Kit (Catalogue # 30–121 1012 K). Early passage primary MEFs were harvested and cultured from C57BL/6 mice as previously described [20 (link), 44 (link), 45 (link)]. β-gal staining was achieved using the Cellular Senescence Kit (Millipore). Recombinant proteins included Cyclophilin A (R&D) and S100A4 (Abcam). SN52 peptide (AAVALLPAVLLALLAPVQRKRRKALP) and SN52-mut peptide (AAVALLPAVLLALLAPVQRNGRKALP) were acquired form GenScript. Inhibitors used were: WP1066, BMS345541, G06976, Staurosporine, and KU60019 (all from Selleck), and Roscovitine (Sigma). The following siRNAs were used: siGENOME non-targeting siRNA#3 (Dharmacon) and si-STAT3 (sc-29,493, Santa Cruz). siRNA transfection was performed with Oligofectamine (Invitrogen).

In this experiment, siRNA was used to knock down the expression of STAT3 mRNA in ESCC cells. Specifically, si-STAT3 (sc-29493, Santa Cruz Biotechnology, Dallas, TX, USA) was used as the specific siRNA targeting STAT3 mRNA, while MISSION^® siRNA Universal Negative Control #1 (NC, Sigma-Aldrich) was used as a negative control. Lipofectamine RNAiMAX (Invitrogen) was used to transfect siRNAs into the ESCC cells. The cells were then cultured in RPMI-1640 medium supplemented with 10% FBS for 2 days before the culture media containing siRNAs were removed. The cells were then passed to be halved in their numbers and cultured in RPMI-1640 without FBS for 2 days before being used for PCR and ELISA experiments.

All cytokines and growth factors including TGFβ1 were purchased from Peprotech (Rocky Hill, NJ, United States). Hydroxyproline assay kits, and CCl₄ were purchased from Sigma-Aldrich (St. Louis, MO, United States). Bleomycin and target specific pooled siRNAs siSTAT3 and siSTAT6 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, United States). EPRS in pEXPR-103-Strep vector (IBA Lifesciences, Göettingen, Germany) were gifts from Dr. Myung Hee Kim at the Korea Research Institute of Bioscience and Biotechnology (KRIBB, Daejeon, Korea). EPRS (1–1440 amino acids) consists of ERS (1–687 amino acids) and PRS (935–1,440 amino acids) linked via non-catalytic WHEP repeat domains (688–934 amino acids) (Ray and Fox, 2014 (link)). The PRS domain of EPRS was cloned into pEXPR-103-Strep vector (IBA Lifesciences). pRc/CMV-WT STAT3 was previously described (Choi et al., 2009 (link)) and pCMV-STAT6-IRES-Neo was a gift from Axel Nohturfft (Addgene plasmid # 35482). Adenovirus expressing SMAD2 or SMAD3 were described previously (Lee et al., 2005 (link)).