Tetracyclin

Human osteosarcoma U2OS cells were grown in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 6% FCS (Lonza), 2 mM l-glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin. Cell lines expressing LAP-Plk1, AKAP-LAP-Plk1, H2B-LAP-Plk1, and GFP-Bora under the control of tetracycline-inducible were cultured in DMEM containing Tet system approved fetal bovine serum (Lonza). Antibodies that were used were directed against Plk1 (18 (link), 19 (link)), Plk1, Cyclin B1, Actin (all from Santa Cruz), GFP (Roche), Plk1-pT210 (BD), Tubulin (Sigma), Bora (17 (link)), Aurora A (Cell Signaling) Histone H3-pS10, and H2AX (both from Upstate). The following drugs were used: BI 2536 (100 nM, Boehringer Ingelheim Pharma), MLN8054 (1 μM, Millennium Pharmaceuticals), thymidine (2.5 mM, Sigma), caffeine (5 mM, Sigma), adriamycin (0.5 μM, Sigma), nocodazole (250 ng/ml, Sigma), PI (Sigma) puromycin (Sigma, 2 μg/ml), and tetracyclin (Sigma, 1 μg/ml).

Lund Human Mesencephalic (LUHMES) cells were obtained, maintained and differentiated as previously described[23 (link)]. Briefly, proliferating LUHMES cells were cultured in Advanced Dulbecco’s modified Eagle’s medium/F12 (DMEM/F12, Gibco) supplemented with 1xN2 (Gibco), 2 mM L-glutamine (Gibco) and 40 ng/mL recombinant basic fibroblast growth factor (R&D Systems). For differentiation, proliferation medium was replaced by DMEM/F12 containing 1xN2, 2 mM L-glutamine, 1 mM dibutyryl cAMP (Sigma Aldrich), 1 μg/mL tetracyclin (Sigma-Aldrich) and 2 ng/mL recombinant human GDNF (R&D Systems). Two days (D2) after adding differentiation medium, cells were seeded in plates (Nunclon) pre-coated with 50 μg/mL poly-L-ornithine and 1 μg/mL fibronectin (Sigma-Aldrich), and grown at 37°C in humidified 5% CO₂ atmosphere.

Antibiotics tested were linezolid, fusidic acid, kanamycin, gentamycin, erythromycin, lincomycin, tetracyclin, vancomycin and ciprofloxacin. Erythromycin and fusidic acid were from Koçak Farma (Tekirdağ, Türkiye), kanamycin, tetracyclin and vancomycin were purchased from Sigma, and commercial injectable preparations were used for the remaining antimicrobials. Agar dilution method was used as described previously [5 ]. Shortly plates were prepared with serial dilution from 64 or 128 mg/L antibiotic concentrations. Inoculum with 5X10⁴ bacteria was placed onto agar using multipoint inoculator. After 16–20 h incubation at 37°C the lowest concentration that inhibits bacterial growth was accepted as MIC. Reference strain S. aureus RN4220 was included to each run.

Human mesencephalon neuronal precursor cells (MESC2.10 cells) were cultured in poly-D-lysine (Sigma, St Louis, MO, USA) coated flasks (75 cm²) in Dulbeccos modified Eagle medium (DMEM)/F12 medium (Gibco, Invitrogen, Calrsbad, CA, USA) supplemented with B27 (Gibco) and Penicillin/streptomycin and human basic FGF 20 ng/ml (Peprotech, Rocky Hill, NJ, USA). To induce neuronal differentiation, cells were cultured for 6 days on poly-D-lysine/laminin (Sigma) coated wells at a density of 30,000 cells /cm² in a medium containing 1 μg/ml tetracyclin (Sigma) (Lotharius et al. 2002 (link); Di Liberto et al. 2012 (link)). Medium was changed every second day and cell differentiation was monitored by the expression of markers for dopaminergic neurons such as tyrosine hydroxylase and dopamine transporter. Human dopaminergic cells were treated with 50 ng/ml FGF21 and analyzed as described in the text. In some experiments 20 μM nicotinamide (NAM) was used to the cells to inhibit SIRT1. Glial cultures from newborn rodent brains were prepared and cultured in 10% fetal calf serum in DMEM as described previously (Lindholm et al. 1992 (link); Mäkelä et al. 2010 (link)). The human hepatocyte cell line (Huh7) was used as a positive control for FGF21 expression (Do et al. 2012 (link)).

Fetal bovine serum (FBS), trypsin/EDTA (0.05%/0.02%), ethylene diamine tri acetic acid (EDTA), Dulbecco’s Phosphate Buffered Saline (DPBS), Protease inhibitors cocktail (PI), Tween 20, Tetracyclin, Penicillin–Streptomycin (Pen-Strep) were purchased from Sigma-Aldrich (Munich, Germany). Blasticidin and Hygromycin B were obtained from Invivogen. Tag-Lite® dopamine D2 receptor red antagonist (spiperone—Cy5) was purchased from Cisbio Bioassays. The total protein concentration has been determined using the Pierce™ BCA protein assay kit provided by ThermoFischer Scientific.