Recombinant human macrophage colony stimulating factor m csf

α-MEM and fetal bovine serum were obtained from GIBCO (Gibco Europe, Uxbridge, UK). Ficoll-Hypaque solution was obtained from Hao Yang Bio. (Tianjing, China). Recombinant human macrophage-colony stimulating factor (M-CSF) and nuclear factor-ĸB ligand (RANKL) were purchased from R & D Systems (Minneapolis, MN, USA). Anti-human RAC1, vinculin, β-actin, and integrin a_vβ₃ antibodies were obtained from Abcam (Cambridge, MA, USA). Alexa Fluor 488 goat anti-mouse IgG antibody was obtained from Thermo Fisher Scientific (Waltham, MA, USA). The TRAP staining kit was purchased from Sigma-Aldrich (St. Louis, MO, USA). Rhodamine-conjugated phylloidin for labeling of filamentous actin was obtained from Sigma-Aldrich. Total mRNA was extracted using Trizol reagent (Invitrogen, Waltham, MA, USA). The reagents for reverse transcription and quantitative fluorescence reaction were purchased from TAKARA (Dalian, China).

DMSO, 2 ​μg/ml of Actinomycin D (Act D), 5 ​μg/ml of cycloheximide (CHX), 4 ​mM of aspirin, and 300 ​μM of sulfasalazine, were purchased from Sigma–Aldrich (Miamisburg, OH, USA). Twenty-five ng/ml of human RANKL and 25 ​ng/ml of recombinant human macrophage colony stimulating factor (M-CSF) were purchased from R&D Systems Inc (Minneapolis, Minnesota, USA) to induce osteoclastic differentiation. Three U/μg of RNase R was purchased from Epicentre Technologies (Madison, WI, USA). MG132 (10 ​μM) was purchased from Selleck Chemicals (Houston, TX, USA). Infliximab was purchased from Shanghai Teramabs Biotechnology Co., Ltd (Shanghai, China). Triptolide at 7 ​nM and 14 ​nM were purchased from EFEBIO Co., Ltd (Shanghai, China). Methotrexate was purchased from Minapharm Pharmaceuticals (Cairo, Egypt).

All chemicals used in the experiments were of analytical grade and were purchased from Sigma-Aldrich (St. Louis, MO, USA). Cell culture medium was purchased from Gibco (Gibco-BRL, Grand Island, NY, USA). Anti-DEFA-1 and β-actin were purchased from Novus (Novus Biologicals, Centennial, CO, USA). Recombinant human receptor activator of nuclear factor kappa-B ligand (RANKL) and recombinant human macrophage colony-stimulating factor (M-CSF) were purchased from R&D System (Minneapolis, MN, USA).

The FOP-iPSCs used in this study were established from a FOP patient harboring R206H heterozygous mutation in ACVR1 [16 (link)], and mutation-corrected resFOP-iPSCs were generated by BAC-based homologous recombination [6 (link)]. iPSCs were maintained in StemFit AK02N (Ajinomot) on iMatrix 511 silk (Nippi)-coated dishes.
Monocytes were induced from FOP- and resFOP-iPSCs by a previously described method with some modification [17 (link)] and then immortalized using lentivirus vectors containing BMI1, cMYC, and MDM2 genes in the presence of polybrene (Sigma) [18 (link), 19 (link)]. Immortalized monocyte cell lines (FOP- and resFOP-ML) were maintained in StemPro-34 (Gibco) supplemented with 2 mM L-glutamine (Gibco), 50 ng/mL recombinant human macrophage colony stimulating factor (M-CSF) (R&D Systems), and 50 ng/mL recombinant human granulocyte macrophage colony stimulating factor (GM-CSF) (R&D Systems) [20 (link)]. CD14⁺ FOP- and resFOP-ML were collected by magnetic-activated cell sorting (MACS) using anti-human CD14 MicroBeads (Miltenyi Biotec) every time before use in each experiment, as per the manufacturer’s protocol.

Recombinant human macrophage-colony stimulating factor (M-CSF) and RANKL were purchased from R&D systems (Minneapolis, MN, USA) and PeproTech EC Ltd. (London, UK) respectively. The TRAP staining kit was purchased from FUJIFILM Wako Pure Chemical Corporation (Tokyo, Japan). Lipopolysaccharide (LPS), BCAAs (L-leucine, L-isoleucine, L-valine), and BCKAs [KIC (4-Methyl-2-oxovaleric acid), KMV ((±)-3-Methyl-2-oxovaleric acid sodium salt) and KIV (Sodium 3-methyl-2-oxobutyrate)] were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). Gabapentin was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The following antibodies were used in western blotting: anti-ERK, anti-phospho-ERK (T202/Y204), anti-p38, anti-phospho-p38 (T180/Y182), anti-JNK, anti-phospho-JNK (T183/Y185), anti-AKT, anti-phospho-AKT (S473), anti-p65, anti-phospho-p65 (Y536), anti-IκBα (Cell Signaling Technology, Danvers, MA, USA), anti-β-Actin, anti-NFATc1 (Santa Cruz Biotechnology), anti-BCAT1 (Novus Biologicals, Centennial, CO, USA), anti-BCAT2 (Abcam, Cambridge, UK) and anti-FLAG (Sigma-Aldrich). The anti-Atp6v0d2 antibody was provided by Y. Choi (University of Pennsylvania, Philadelphia PA, USA).

Recombinant human (rh) macrophage colony-stimulating factor (M-CSF) and rh interleukin (IL)-10 were from R&D Systems (Minneapolis, MN, USA). Interferon (IFN)-γ, anti-CD14-coated microbeads, phycoerytrin (PE)-CD163 monoclonal antibody (mAb) and fluorescein isothiocyanate (FITC)-CD206 mAb were from Miltenyi Biotec (Gladbach, Germany). Allophycocyanin (APC)-CD16, APC-Alexa Fluor 750-human leukocyte antigen-D region-related (HLA-DR) and APC-Alexa Fluor 700-CD36, PE-Cyanine dye Cy7 (PE-Cy7)-CD197 (CCR7) mAbs were from Beckman Coulter (San Jose, CA, USA). PE-CD1a and FITC-CD14 mAbs were from PharMingen (San Diego, CA, USA). Fetal bovine serum (FBS) was from Hyclone Laboratories (Logan, UT, USA). RPMI 1640 was from GIBCO (Paisley, UK). Sytox^® Blue dead cell stain and 2-7-dichlorodihydrofluorescein diacetate (H2DCF-DA) were purchased from Molecular Probes (Carlsband, CA, USA). Propranolol, isoproterenol, lipopolysaccharide (LPS; from Escherichia coli strain 0111:B4), trypan blue solution and FITC-dextran were from Sigma-Aldrich (Milan, Italy). ML385, a novel and specific Nrf2 inhibitor, was purchased from Selleck Chemicals (Cologne, Germany).

Recombinant human (rh) macrophage colony-stimulating factor (M-CSF) was purchased from R&D System (Minneapolis, MN). Fetal bovine serum (FBS) was purchased from Hyclone Laboratories (Logan, UT). Anti-CD14-coated microbeads, human IFN-γ1b (IFN-γ), fluorescein isothiocyanate- (FITC-) conjugated mAbs to human leukocyte antigen-D region related (HLA-DR), and VioGreen-conjugated mAbs to CD36 were purchased from Miltenyi Biotec (Gladbach, Germany). Lupeol, 7KC, phorbol 12-myristate 13-acetate (PMA), lipopolysaccharides from Escherichia coli (LPS), FITC-dextran, and 2′,7′-Dichlorofluorescein Diacetate (H2DCF-DA) were purchased from Sigma-Aldrich (Milan, Italy). Sytox Blue nucleic acid stain and 4′,6-diamidino-2-phenylindole (DAPI) were purchased from Thermo Fisher Scientific (Waltham, Massachusetts, USA).