Leflunomide

¹⁵N-labelled glutamine (Cambridge Isotope Laboratories NLM-557-0.5), D-glucose (Sigma G7528), L-glutamine (Sigma G3126), AZD8330 (Selleckchem, S2134), trametinib (Selleckchem, S2673), GDC0941 (Selleckchem, S1065), leflunomide (Sigma L-5025), brequinar sodium salt (Sigma, SML0113), uridine (Sigma, U3003), inosine (Sigma, I4125), mitochondrial stress kit (Seahorse 101706-100), VECTASTAIN Elite ABC Kit (pk-6100; Vector Labs), DAB (sk-4100; Vector labs).

Collagen Type II was prepared as previously described from bovine cartilage [35] . Complete Freund Adjuvant (CFA), leflunomide, hexadecyl trimethylammonium bromide (HTAB), and o-dianisidin were purchased from Sigma-Aldrich Chemical Company (St. Louis, MO, USA). Nimesulide was gifted from Alkan Pharmaceutical Co., (6^th of October city, Egypt).

Prior to library screening, the reliability of the screening method was confirmed using known osteogenic inducible bioactive factors, harmine (Biomol, Plymouth Meeting, PA, USA) [16 (link)], phenamil (Sigma) [17 (link),18 (link)], resveratrol (Sigma) [19 (link),20 (link),21 (link),22 (link)], and recombinant human BMP2 [23 (link),24 (link),25 (link)] (Peprotech, London, UK) as positive controls. After library screening, the candidates for osteogenesis targeting, leflunomide (Lef), 1-(5-isoquinolinylsulfonil)-3-metylpiperazine dihydrochloride (1-5), and LFM-A13 (LFM), were purchased from Sigma, Santa Cruz Biotechnology (Dallas, TX, USA) and Calbiochem (Beeston Nottingham, UK), respectively, to confirm their abilities.

XBJ was obtained from Tianjin Chase Sun Pharmaceutical Co. Ltd. (Tianjin, China) with the lot number of 1204181. Thioglycollate broth, lipopolysaccharide (LPS), and leflunomide were purchased from Sigma-Aldrich (St. Louis, MO). Allophycocyanin- (APC-) anti-CD11c and fluorescein isothiocyanate- (FITC-) anti-CD206 were purchased from BioLegend (San Diego, CA). Phycoerythrin- (PE-) anti-F4/80 was purchased from eBioscience (San Diego, CA). Mouse TNF-α, IL-6, and IL-10 enzyme-linked immunosorbent assay (ELISA) kits were purchased from Excel Bio company (Shanghai, China). JAK1 and STAT6 fast activated cell-based ELISA (FACE) kits were purchased from Active Motif (Carlsbad, CA). Mouse anti-Arg1 was purchased from BD Biosciences (San Jose, CA), mouse Fizz1 antibody was purchased from R&D Systems (Minneapolis, Minn), anti-Ym1 was purchased from Stemcell Technologies (Vancouver, Canada), and anti-β-actin was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). JAK1 inhibitor was purchased from Calbiochem (San Diego, CA).

Quercetin was purchased from Sigma-Aldrich Co., Ltd. (St. Louis, MO, USA) as a preservative-free pure powder. It was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10.0 mM and was then diluted with Airway Epithelial Cell Growth Media (AECG medium; PromoCell GmbH, Heidelberg, Germany) at appropriate concentrations for the experiments, sterilized by passing through 0.2 μm filters, and stored at 4°C until use. Recombinant human IL-4 was purchased from R & D Systems, Inc. (Minneapolis, MN, USA), as a preservative-free pure powder. IL-4 was also dissolved in an AECG medium, then sterilized, and stored at 4°C until use. Leflunomide, which is a Janus kinase (JAK)-STAT6 inhibitor [27 (link), 28 (link)], was purchased from Sigma-Aldrich Co., Ltd., and was first dissolved in DMSO at a concentration of 1.0 mg/ml. This was followed by dilution in an AECG medium at appropriate concentrations for the experiments. mRNA isolation kits were purchased from Milteny Biotec (Bergisch Gladbach, Germany). The reagents used for the cDNA synthesis and quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR; TaqMan Gene Expression Assays) kit were purchased from Invitrogen Corp. (Carlsbad, CA, USA) and Applied Biosystems (Foster City, CA, USA), respectively.

Mdivi-1 (Sigma), an inhibitor of mitochondrial fission, was dissolved in dimethylsulfoxide (DMSO) and stored at −20°C before use, and fresh medium was used to obtain a final concentration of 30 μM. Leflunomide and teriflunomide (Sigma), two different activators of mitochondrial fusion, were dissolved in DMSO at appropriate concentrations. Prior to cell treatment, fresh medium was used to obtain a concentration of 75 μM for both drugs. After the cells were fully attached to the PDMS membrane in the cell culture channel, medium containing a mitochondrial fusion activator (Leflunomide and teriflunomide) or mitochondrial fission inhibitor (Mdivi-1) was added to the aorta smooth muscle-on-a-chip models. After 24 hr of rhythmic strain, the samples were collected for comparative experiments.