F 12 coon s modification medium

Rat thyroid follicular cells PCCL3 (16 (link)) were used to establish stable cell line PCCL3-FAM83F by transfecting pCMV-neo-FAM83F (RC203874) containing Myc-DDK-tagged human FAM83F coding sequence (Origene Technologies, Rockville, MD). PCCL3-empty control cells (PCCL3-Ø) were established with empty plasmid and G418 selection. These cells were cultivated in F-12 Coon‘s Modification medium (Sigma, St. Louis, MO, USA) supplemented with 5% fetal bovine serum (FBS) and four hormones: 1 mU/ml bovine TSH (Sigma), 10 μg/ml insulin (Sigma), 5 μg/ml transferrin (Sigma), and 10 nM hydrocortisone (Sigma), 300 ug/ml G418 (Invitrogen, Carlsbad, CA).
Nthy-ori 3-1 derived from normal human primary thyroid follicular epithelial cells were purchased from ECAAC (European Collection of Cell Culture) and cultivated RPMI (Invitrogen) supplemented with 2 mM l-glutamine and 10% FBS. All cell lines were kept in humidified incubator at 37°C and 5% CO₂.

Quercetin dihidydrate (QU, 98% purity) was purchased from Fluka, BioChemica, Switzerland. The anticancer drug cisplatin [cis-diamindikloroplatinum(II)] was supplied by Pliva, Zagreb, Croatia. F-12 Coon’s Modification medium, MTT [3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide] for the evaluation of cell viability and proliferation, and fetal calf serum (FCS) were purchased from Sigma-Aldrich, St. Louis, MO, USA.

Yeast cells from a P. brasiliensis-virulent strain (Pb18) were cultured for 7 days at 37°C in brain heart infusion (BHI)-agar medium (Sigma) supplemented with gentamicin (96 μg/mL), and 5% fetal bovine serum (FBS) (Gibco). Pb18 cells were harvested and incubated overnight under agitation in F12 Coon's Modification medium (Sigma) at 37°C. Cell viability was determined by the fluorescein diacetate-ethidium bromide method, as previously described [104 (link)]. Only fungal suspensions containing more than 90% viable cells were used. For in vivo infection, six to seven-week-old male mice were anesthetized with ketamina (90mg/kg) and xylazine (5mg/kg) by intraperitoneal (i.p.) administration, followed by intravenous (i.v.) inoculation of 1x10⁶ yeast cells. The numbers of colony-forming units (CFU) in the organs were calculated at 15 and 30 days post-infection (dpi) by normalizing the count per gram of tissue. The survival of the Pb18-infected wild-type and knockout mice (10–12 mice of each group) was verified daily for 120 days.