Interferon gamma ifn γ

The protocol for the culture and differentiation of MSC into osteogenic or adipogenic lineages were described in the Supplementary Methods. Briefly, MSC were plated onto 6 well plates at a concentration of 1 × 10⁴ cells/cm² and adhered for 2 days. After that the cells were washed twice with phosphate buffered saline and incubated in culture medium with or without stimulating agents for 24 h with proinflammatory cytokines interferon-gamma (IFN-γ; 500 U/mL) (R&D Systems, Minneapolis, MN) and tumour necrosis factor-alpha (TNF-α; 50, 500 and 5000 U/mL) (R&D Systems) alone or by a proinflammatory cytokine cocktail of IFN-γ (500 U/mL) and TNF-α at varied concentrations of 50, 500 and 5000 U/mL.

Human CRC cell lines (HCT116, HT29, LoVo, and SW48), a human monocyte cell line (THP-1), and a mouse colon cancer cell line (CT26) were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured in Rosewell Park Memorial Institute (RPMI) 1640 (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin–streptomycin (Gibco, NY,USA) in a 5% CO₂ incubator at 37 °C. According to previous studies^{28 (link),29 (link)}, to polarize M0 macrophages, THP-1 cells (5 × 10⁶) were plated in 100-phi dishes and stimulated with 320 nm phorbol 12-myristate 13-acetate (PMA; Sigma, St. Louis, MO, USA), followed by incubation at 37 °C for 24 h. The culture supernatant was collected and labeled M0 macrophage-conditioned medium (M0 CM). To polarize M1 or M2 macrophages, THP-1 cells were treated with PMA for 6 h, and M1-polarizing reagents (100 ng/ml lipopolysaccharide (LPS) plus 20 ng/ml interferon gamma (IFN-γ); R&D, Waltham, MA) or M2-polarizing reagents (20 ng/ml IL-4 plus 20 ng/ml IL-13) were added, followed by incubation at 37 °C for 18 h. The culture supernatant was collected and labeled M1 macrophage CM (M1 CM) or M2 macrophage CM (M2 CM). MG132 (proteasome inhibitor) and PI3Kγ inhibitors (TG100-115 and IPI-549) were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Selleckchem (Houston, TX, USA), respectively.

THP-1 cells, constituting a human leukemic cell line, were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and grown in RPMI 1640 medium with 2 mM l-glutamate, 4.5 g/L glucose, 10 mmol/L HEPES, 1.0 mmol/L sodium pyruvate, 10% fetal bovine serum (FBS), and 1% antibiotic antimycotic mixture. Cell density was maintained between 5 × 10⁴ and 8 × 10⁵ viable cells/mL, and the medium was refreshed every 2–3 days. THP-1 monocytes were differentiated into macrophages through 24 h incubation with 100 nM phorbol 12-myristate 13-acetate (PMA) (Sigma, St. Louis, MO, USA). Macrophages were polarized to M1 macrophages through addition of 100 ng/mL lipopolysaccharide (LPS) (Sigma-Aldrich) and 20 ng/mL Interferon gamma (IFN-γ) (R&D Systems). Cells were then washed with PBS twice and cultured with RPMI 1640–1% FBS for 48 h. The conditioned media (CM) were harvested and filtered through a 0.22-μm filter to remove cell debris to obtain THP-1 CM, CON siRNA + M1 CM and Gal-3 siRNA + M1 CM, and stored in aliquots at −20 °C. The A7r5 cells, the rat aortic SMCs, were purchased from the ATCC and were expanded in DMEM supplemented with 2 mM glutamine and 10% FBS at 37 °C in an incubator perfused with 5% CO2 and 95% O2. Prior to experimentation, the A7r5 cells were exposed to CM for the subsequent studies.