Murine macrophage colony stimulating factor m csf

Lipopolysaccharide (Escherichia coli 0111:B4) was purchased from Sigma. CL264 and Poly I:C were purchased from InvivoGen. Murine macrophage colony-stimulating factor (M-CSF) was obtained from PeproTech. Anti-Blimp-1 antibody, anti-IRF5, anti-TRAF2 antibodies were obtained from cell signaling technology. Anti-β-actin antibody was obtained from Santa Cruz.

For BMDM, bone marrow was collected by flushing the femurs and tibia of C57BL/6 mice (6–8 weeks old) with cold PBS. After collection, red blood cells (RBC) were lysed with RBC lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA), and the remaining cells were washed twice with cold PBS. To induce macrophage differentiation, bone marrow cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) and 20 ng/mL murine macrophage colony-stimulating factor (M-CSF) (PeproTech, USA) for 6 days. Fresh medium with M-CSF was added every 3 days. After polarization, the cells were phenotyped and used in different assays. For pMAC, C57BL/6 mice were intraperitoneally injected with 1 mL of 3% thioglycollate medium. After 3 days, pMAC were isolated from the peritoneal cavity of the injected C57BL/6 mice by flushing the peritoneal cavity with 5 mL of ice-cold PBS. The cells were centrifuged and resuspended in RPMI-1640 (12633; Gibco) supplemented with 10% FBS for further assays. For M2-like polarization, BMDM on day 7 or pMAC were treated with 20 ng/mL murine IL-4 and 20 ng/mL IL-13 (PeproTech, USA) for 24 h.

L1210 murine leukemia cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS), 2 mM l-glutamine, 100 µg/mL streptomycin, and 100 U/mL penicillin. Cells were maintained in 5% CO₂ to 95% air. To culture bone marrow-derived macrophages (BMDMs), mononuclear cells were isolated from tibias and femurs of C57BL/6 mice. Cells were cultured at a density of 2 × 10⁶/mL in DMEM containing 10% FBS and 25 ng/mL murine macrophage-colony stimulating factor (M-CSF) (PeproTech, Rocky Hill, NJ, USA) for 7 days. In some experiments, IFNγ (20 ng/mL, PeproTech), LPS (50 ng/mL, Sigma, St. Louis, MO, USA), or IL4 (20 ng/mL, PeproTech) was added and cultured for 24 h before further analyses. Macrophages treated with PBS, LPS + IFNγ, or IL4 were named as M^PBS, M^LPS, or M^IL4, according to Epelmann et al. (5 (link)). Cells were transfected siRNA with Lipofectamine LTX (Invitrogen) according to the recommended protocol. Three pairs of siRNA for each target were designed and their knockdown efficiency was determined by qRT-PCR and Western blotting. The siRNA with highest efficiency was chosen for further experiments. Lentivirus was packaged by Cyagen Biosciences with commercial service (Guangzhou, China).

Medium, supplements, and fetal bovine serum (FBS) for cell culture were purchased from GIBCO BRL (Grand Island, N.Y.). Small molecule inhibiting agents: ALK5 inhibitor SB431542, PI3K inhibitor LY294002, NF-κB inhibitor BAY11-7082, ERK inhibitor PD98059 were obtained from Beyotime. Lipopolysaccharide (LPS), phorbol-12-myristate-13 acetate (PMA) and FITC-latex beads were purchased from Sigma-Aldrich (St Louis, MO). Primary antibodies against SNAIL, p-Akt (Ser473), Akt, p-Smad2, Smad2, p-Erk1/2, p-β-catenin, p-IκBα were purchased from Cell Signaling Technology (MA, USA) and anti-β-catenin, β-actin, α-Tubulin, GAPDH were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). FITC- or PE-conjugated antibodies for flow cytometry were supplied as follows: F4/80, CD80, CD86, from eBioscience (San Diego, CA); CCR7, CD206 from BD Biosciences (San Jose, CA). Lipofectamine 2000 was purchased from Invitrogen (Carlsbad, CA, USA). Recombinant protein human and mouse TGF-β1, human IFN-γ, and murine macrophage colony stimulating factor (M-CSF) were bought from Pepro Tech (Rocky Hill, NJ). PrimeScript^® RT reagent Kit and SYBR^® Premix Ex Taq™ were products of TaKaRa. E.Z.N.Z^® HP Total RNA Kit was bought from Omega Bio-Tek (Norcross, CA, USA).