Sc 8019

MIA-PaCa 2 cells were seeded in a 6-well tissue culture plate (cell density 1.5 × 10⁶/well) in DMEM medium supplemented with 10% foetal bovine serum, 1% L-glutamine, and 1% penicillin and streptomycin at 37 °C and 5% CO₂. Cells were serum-starved for 24 h and then incubated with LIF (10 ng/mL) alone or plus mifepristone (10, 20, 50 µM) for 10 min. Total lysates were prepared by homogenization of MIA-PaCa2 cells in Ripa buffer containing phosphatase and protease inhibitors. Protein extracts were electrophoresed on 12% acrylamide Tris-Glycine gel (Invitrogen), blotted to the nitrocellulose membrane, and then incubated overnight with primary Abs against STAT3 (sc-8019 1:500; Santa Cruz Biotechnology) and phosho-Stat3 (GTX118000 1:1000; Genetex). Primary Abs were detected with the HRP-labelled secondary Abs. Proteins were visualized by Immobilon Western Chemiluminescent Reagent (MilliporeSigma) and iBright Imaging Systems (Invitrogen). Quantitative densitometry analysis was performed using ImageJ software. The degree of STAT3 phosphorylation was calculated as the ratio between the densitometry readings of p-STAT3/STAT3.

Immunohistochemical analysis was done in all animals included in each group. Primary Abs were the monocyte/macrophage marker CD68 (Ab53444; Abcam), the smooth muscle cell marker smooth muscle actin (Clone 1A4; Sigma), calponin (ab46794; Abcam) and osteopontin (ab216406; Abcam), phospho-STAT1 (33–3400, ThermoFisher Scientific), phospho-STAT3 (sc8019, Santa Cruz Biotechnology) and ICAM-1 (ab124760, Abcam).
Donkey anti-goat biotin, sheep anti-mouse biotin, and goat anti-rat biotin (Amersham) were used as secondary antibodies. ABComplex/HRP was then added and sections were stained with AEC substrate-chromogen (DAKO), counterstained with hematoxylin, and mounted in Pertex (Medite). Incubation without primary Abs and/or irrelevant species- and isotype-matched immunoglobulins was used as a negative control for all immunostainings. For colocalization studies anti-rabbit Alexa Fluor 488 and anti-rat Alexa Fluor 568 were used as secondary Abs.
Computer-assisted morphometric analysis was performed with the Image-Pro Plus software (version 1.0 for Windows). The threshold setting for area measurement was equal for all images. Samples from each animal were examined in a blinded manner. Results were expressed as % positive area versus total area (collagen, macrophages, α-actin, calponin, osteopontin and ICAM-1), or positive nuclei per mm² (p-STAT-1 and p-STAT3). Faltan calponin and osteopontin

A ChIP assay was performed using a SimpleChIP Plus Kit (Cell Signaling Technologies (CST), Beverly, MA, USA) according to the kit's instructions. In short, 2 × 10⁶ HEK-293T cells were cultured in 10 cm dishes. Cells were crosslinked using 1% methanol, and the reaction was terminated by glycine. The chromatin was detached in micrococcal nuclease. Anti-IgG (negative control (NC), mouse monoclonal IgG (CST) and anti-STAT3 (1 : 1,000, sc-8019, Santa Cruz Biotechnology) were reacted with the Protein G magnet beads to form protein-nucleic acid complexes (immunoprecipitates). The chromatin precipitates were eluted and purified, and the enrichment of STAT3 fragments was quantified using qPCR.

To analyze the IL-6-induced phosphorylation of transcription factors, LF fibroblasts grown in a 6-well plate were incubated for 15 min with IL-6 (300 ng/ml) and lysed in radioimmmunoprecipitation assay buffer (Nacalai Tesque). The lysates were homogenized in sample buffer solution containing 2-mercaptoethanol (2×) (Nacalai Tesque) and proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane (Invitrogen) that was blocked in blocking reagent (Block-Ace; DS Pharma Biomedical, Osaka, Japan) for 30 min at 25 °C and treated overnight at 4 °C with the following primary antibodies: anti-human STAT3 (mouse monoclonal, 1:500, SC-8019) and p-STAT3 (1:500) (both from Santa Cruz Biotechnology); and anti-human ERK1/2 (mouse monoclonal, 1:1000, AB54230), p-ERK1/2 (1:1000), p38 (rabbit monoclonal, 1:1000, AB170099), p-p38 (1:1000), JNK (rabbit polyclonal, 1:500, AB112501), and p-JNK (1:500) (all from Abcam). The membrane was then incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (Santa Cruz Biotechnology) for 1 h at 25 °C and immunoreactivity was visualized with Chemi-Lumi One Super (Nacalai Tesque) and a luminescent image analyzer (EZ-capture2; ATTO, Tokyo, Japan) and quantified using ImageJ v.1.47 software (National Institutes of Health, Bethesda, MD, USA).