Human il 13

The metastatic KM12SM colon cancer cell line was acquired from Dr. I. Fidler (MD Anderson Cancer Center, Houston, TX, USA). The cell lines SW480 and RKO were obtained from the ATCC and the SW620 cell line from the European Collection of Authenticated Cell Cultures (Salisbury, UK). HT-29 colon cancer cells were a kind gift from Dr. Mollinedo (CIB-CSIC). KM12SM were authenticated in our laboratory every 6 months. Murine CT26 colon carcinoma cell lines were obtained from Prof. Caroline Saucier (Université de Sherbrooke, Sherbrooke, QC, Canada). All cell lines were cultured as previously described [19 (link)]. Human IL-13 was used at 10 ng/mL and was purchased from PeproTech (London, UK). Irinotecan (Selleckchem, Munich, Germany) was used at 50 mg/Kg of weight. Antibodies used in the experiments are listed in Table S1.

THP-1 cells and PBMCs were used to generate M2 macrophages. For macrophage differentiation, THP-1 cells were treated overnight with 200 nM phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich, St. Louis, MO, USA) in RPMI 1640. PBMCs were treated with 30 ng/ml of recombinant human macrophage colony-stimulating factor (M-CSF; Peprotech) for 6 days. Half of the medium was replaced with fresh medium containing M-CSF on day 3. To induce M2 macrophages, THP-1 cells were further treated with 25 ng/ml human interleukin (IL)-4 and 25 ng/ml human IL-13 (both from Peprotech) for 48 h.

All experiments were performed with the approval of the Ethics Committee and in accordance with the guidelines of the Institutional Animal Care and Use Committee of Gyeongsang National University (GNU-160811-M0036). Unless otherwise stated, all chemicals were purchased from Sigma-Aldrich (St Louis, MO, USA). Stock solutions of colivelin (378 µM; Tocris Bioscience, Bristol, UK), doxorubicin (2 mg/mL), and lipopolysaccharide (LPS; 5 mg/mL) were prepared in distilled water. The mouse IL-4 (0.1 mg/mL), human IL-4 (0.1 mg/mL), human IL-13 (0.1 mg/mL), and human IFN-γ (0.1 mg/mL), which were purchased from PeproTech (Rocky Hill, NJ, USA), were dissolved in distilled water. Bay 11-7085 (50 mM), necrostatin-1 (20 mg/mL; Cayman Chemical, Ann Arbor, MI, USA), PMA (1 mM), SB203580 (25 mM), S3l-201 (50 mM; Selleckchem, Houston, TX, USA), Z-VAD-FMK (10 mg/mL; InvivoGen, San Diego, CA, USA), and Z-YVAD-FMK (20 mM; Abcam, Cambridge, MA, USA) were dissolved in dimethyl sulfoxide (DMSO). U-46619 (28.53 mM) was prepared in methyl acetate. The solutions were then diluted in culture medium to the working concentration. An equivalent concentration or volume of solvents was added in the control group. The final concentrations of DMSO and methyl acetate were ~0.1% (v/v).