Fludarabine

STAT1 inhibitor Fludarabine, STAT5 inhibitor Ac-4-130, mTORC1 inhibitor rapamycin and Brefeldin A were purchased from MedChemExpress. Nile tilapia was i.p. injected with 5 mg/kg Fludarabine or 1 mg/kg rapamycin every two days during E. piscicida infection. Nile tilapia was i.p. injected with 13 mg/kg Brefeldin A to block cytokine secreting at 6 h before sacrifice, and the spleen leukocytes were isolated for IFN-γ producing by flow cytometry or western blot on day 5 or 8 post infection. For the STAT5 inhibition, the isolated spleen leukocytes were treated with 5 mM Ac-4-130, and the cell were stimulated with or without recombinant IL-2 for 12 h.

The human androgen-independent prostate cancer cell line C4-2B (American Type Culture Collection, VA, United States) was validated by short tandem repeat DNA fingerprinting with an AmpFLSTR Identifiler Kit (Applied Biosystems, CA, United States) at MD Anderson’s Characterized Cell Line Core Facility. The cells were cultured in DMEM containing 1 mM sodium pyruvate, 2.5 mM glutamine, 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C in a humidified incubator containing 5% CO₂. The serum-free medium was regularly used in the cell transfection trials. Enzalutamide (AR inhibitor), AMD3100 (CXCR4 inhibitor), and AZD1480 (ATP competitive antagonist of JAK2) were purchased from SelleckChem, and SDF-1 (CXCR4/7 activated ligand) was purchased from GenScript. CCX771 (CXCR7-specific inhibitor) and CCX704 (the control inhibitor of CXCR7, with the molecular structure similar to CCX771 but not binding to CXCR7) were provided by ChemoCentryx (CA, United States). Fludarabine (STAT1 synthesis inhibitor) and GSI-XII (Notch-1 inhibitor) were obtained from MedChemExpress. In addition, C4-2B cells were seeded at desired densities (5 × 10⁵/well in a six-well plate for western blotting and wound-healing assay; 1 × 10⁵/well in a 24-well plate for flow cytometry; and 1.5 × 10⁴/well in a six-well plate for ELISA).

IFI35 expression was detected in HK-2, ACHN, Ketr-3, and 786-O cell lines. pSTAT1 or pSTAT6 expression was examined in Ketr-3 and 786-O cells transfected with IFI35 shRNA and control shRNA in the presence or absence of the signaling inhibitor Fludarabine (5 μmol/L; MedChem Express, Monmouth Junction, NJ, USA) or AS1517499 (100 nmol/L; MedChem Express) for 24 h. For intracellular staining, cells were washed and fixed with fixation buffer (BD Biosciences) for 20 min at 4 °C. The fixed cells were permeabilized with permeabilization solution (BD Biosciences) at room temperature for 30 min. Cells were incubated with PE-anti-human pSTAT1 (BioLegend, San Diego, CA, USA), PE-anti-human pSTAT6 (BioLegend) in PBS containing 2% FBS for overnight at 4 °C. Isotype-matched anti-human IgG served as a control. Subsequently, the labelled cells were analyzed using a BD FACSCanto II flow cytometer with FACSDiva software (BD Biosciences).

CCK-8 assay was used to measure the viability of Huh7 and HL7702 under treatment of Fludarabine ( Med Chem Express, Shanghai, China). The cells were resuspended and seeded in a 96-well plate (6 × 10⁴ cells/well) and cultured at environment of 37 °C. Each well was added with 10ul cck-8 solution (Yeasen, Shanghai, China) and the proliferation ability were represented with the absorbance at 450 nm tested by Microplate Reader (Bio-Rad, Hercules, CA).

For quantification of the cytostatic effect of IFN-γ, YUMM3.3 cells were seeded at a density of 5x10³ cells/well in 96-well plate and incubated 48 hrs with 20 ng/ml IFN-γ (485-MI, R&D) and 50 ng/ml Activin-A (R&D Systems) or 10 μM SB-431542, and, where indicated, Ruxolitinib or Fludarabine, 0.5 μM Nifuroxazide (all from MedChem Express), 100 μg/ml Carboplatin (C2043-1G, TCI America), E64 (HY-15282, MedChemExpress), or Petesicatib (HY-109069, MedChemExpress). Alamar Blue reagent (Invitrogen, DAL1025) was added to subconfluent cells (<90% confluence, inspected manually), and fluorescence was measured 3-4 hrs later on a TECAN spectrophotometer at the emission wavelength of 590 nm after excitation at 560 nm.

A human gastric mucosal epithelial cell line (GES-1) was purchased from the Xiangya Experiment Center (Changsha, China), Human GC cells (AGS, BGC-823, MGC-803, and SGC-7901) and HEK293 cell lines were purchased from the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Beijing, China). AGS, BGC-823, MGC-803, and SGC-7901 cells were cultured in RPMI 1640 medium (Gibco, California, USA), GES-1 and HEK293 cells were cultured in DMEM (Gibco) with 10% fetal bovine serum (BD Bioscience, San Jose, CA, USA or ExCell Bio, Shanghai, China), at 37 °C in a humidified incubator with 5% CO₂. Culture flasks were purchased from JET BIOFIL (Guangzhou, China). Cells were treated with 2 mg/ml actinomycin D (Meilune, Dalian, China) for 4, 8, 12, and 24 h, with dimethylsulfoxide as a negative control. LSECtin or STAT1 overexpression plasmids, short interfering RNAs (siRNAs) targeting STAT1 or circFBXL4, and miR146a-5p mimic and inhibitor were purchased from GenePharma (Shanghai, China) and then were transfected with Lipofectamine 2000 (Invitrogen, Carlsbad, CA). The sequences and NCBI identifiers are available as Additional files 1 and 2. Cells were treated with the STAT1 activation inhibitor fludarabine (5 µM, 24 h) (MedChemExpress, Dalian, China), with dimethylsulfoxide as a negative control.

MDK antibody (1:1000 dilution, 11009-1-AP) was purchased from Proteintech Group Inc (Rosemont, IL, USA). E-cadherin antibody (1:1000 dilution, 3195T), ZO1 antibody (1:1000 dilution, 8193T), Vimentin antibody (1:1500 dilution, 5741T) and Snail antibody (1:500 dilution, 3879T) were from Cell Signaling Technology (Danvers, Massachusetts, USA). STAT1 antibody (1:5000 dilution, ab109320) and phospho-STAT1 (phosphor Y701) antibody (1:1000 dilution, ab109457) were purchased from Abcam (Cambridge, UK). β-actin antibody (1:1000 dilution, AF5001) was from Beyotime Biotechnology (Shanghai, China). Recombinant human IFN-γ was acquired from PeproTech (Rocky Hill, NJ, USA) and used at a concentration of 50 ng/ml for 48h. The MDK inhibitor iMDK was bought from Merck Millipore (Darmstadt, Germany) and diluted to a final concentration of 100nM in growth mediums. The STAT1 inhibitor Fludarabine was purchased from Med Chem Express (New Jersey, USA) and used at a concentration of 5 μM for 48h.