Teriflunomide

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.

Phagocytic activity of microglia was determined by measuring the uptake of fluorescent latex beads on flow cytometry as previously described [18 (link)]. After 24 h teriflunomide (±12 h LPS; 100 ng/ml; Sigma-Aldrich, Munich, Germany) treatment, FITC-labeled latex beads (1 μm, Fluoresbrite™ Yellow Green carboxylate microspheres; Polysciences, Warrington, USA) were added at a cell:bead ratio of 1:100 and incubated for 1 h at 37 °C. In parallel, cells incubated with beads on ice (4 °C) served as negative controls. Non-phagocytosed and surface bound beads were removed by washing six times with ice-cold PBS. Adherent microglia were then harvested by 0.05 % trypsin/EDTA treatment, and the uptake of the beads was determined by flow cytometry (FACSCalibur; Becton-Dickinson, San Jose, CA, USA). Dead cells were excluded by propidium iodide staining.
A shift in mean fluorescence intensity (MFI) resulting from the uptake of fluorescent beads and the percentage of gated microglia that phagocytosed latex beads were used as a measure to assess phagocytosis. Active phagocytosis was then calculated by subtracting measured values of cells incubated at 4 °C from the values obtained at 37 °C.

D4 LCLs were plated at 2 × 10⁵ cells/ml on day 0 and harvested on day 7. One hour after plating, cells were treated with dimethyl sulfoxide control (DMSO; Sigma-Aldrich); or with 2.5 to 70 μg/ml teriflunomide (A771726; CalBiochem in Figure 1A; ENZO Life Sciences in all others) dissolved in DMSO. The final DMSO concentration in control and treatment groups was 0.1%. uridine-treated cells were given 150 μM uridine one hour prior to teriflunomide or DMSO treatment. Growing cells were expanded into fresh medium containing DMSO or teriflunomide, with or without 150 μM uridine (Sigma-Aldrich), as needed. Cells treated with teriflunomide in Figure 1A were given fresh drug every 24 hours. Cells were counted using trypan blue exclusion (Figure 1A) or relative cell titers were determined using Cell Titer Glo as instructed by the manufacturer (Promega; Figure 1B).

MEDS433 and brequinar were synthesized as described in [4 (link)]. ATRA, Teriflunomide, Dilazep and uridine were purchased from Sigma-Aldrich. Reagents were dissolved in DMSO and diluted in culture medium before use. Final DMSO concentration did not exceed 0.1%. Dilazep was dissolved in water. Ara-C (Citarabina Hospira, Lake Forest, IL, USA), Idarubicin (Zavedos, Pfizer, New York, NY, USA), Dipyridamole (Persantin, Boehringer Ingelheim, Ingelheim am Rheim, Germany), Decitabine (Dacogen, Janssen-Cilag, Beerse, Belgium) were purchased.

Leflunomide, teriflunomide, α-, β- and γ-cyclodextrins were purchased from Sigma-Aldrich. All other reagents (HCl, KH₂PO₄ and Na₂HPO₄⋅12H₂O) used for the preparation of the buffers were of analytical grade. The pH of buffer solutions was controlled using Mettler Toledo Five Easy pH-meter.

The reference standards of anti-androgen compounds (purity ≥ 98%), such as abiraterone acetate, bicalutamide, flutamide, nilutamide, teriflunomide, leflunomide and ailanthone, were purchased from Sigma-Aldrich (Beijing, China). The standards (purity ≥ 98%) of blood urea lowering agents, i.e., allopurinol, oxypurinol and febuxostat, were obtained from Sigma-Aldrich (Rehovot, Israel). The studied compounds were dissolved in DMSO or in ethanol, respectively, to obtain a concentration of 5 mg/mL. The stock solutions were stored at 2–8 °C prior to analyses.