Hff 1

T. gondii parasites (RH Δhxgprt unless otherwise indicated) were grown on human foreskin fibroblasts (HFF-1; purchased from ATCC SCRC-1041), transfected and selected as previously described [22 (link),42 (link)]. Plaque assays were performed as previously described [18 (link)].

Biological activity of the tested compounds was assessed in vitro using cultured cell lines: melanoma C-32 (ATCC, Rockville, MD, USA), glioblastoma SNB-19 (DSMZ, Braunschweig, Germany), breast cancer MDA-MB-231 (ATCC, Rockville, MD, USA), and normal human fibroblasts derived from foreskin HFF-1 (ATCC, Rockville, MD, USA). Cell cultures were maintained using DMEM (Lonza, Basel, Switzerland) supplemented with 10% fetal bovine serum (FBS) (Biological Industries Cromwell, CT, USA) and a mixture of penicillin (10,000 U/mL) and streptomycin (10 mg/mL) (Lonza, Basel, Switzerland).

HEK293 cells (ATCC, # CRL-1573), RAW 264.7 (ATCC, # TIB-71), NCl-H596 cells (ATCC, HTB-178), HFF-1 (ATCC, #SCRC-1041), L929 (ATCC, # CRL-6364), MDA-MB-231 (Sigma, 92020424-1VL), and Vero cells (ATCC, # CCL-81) were maintained in Dulbecco’s Modified Eagle Medium (DMEM) (Life Technologies, # 11995-065) containing antibiotics (Life Technologies, # 15140-122) and 10% fetal bovine serum (FBS) (Life Technologies, # 26140-079). NCI-H1299 cells (ATCC, # CRL-5083) and THP-1 cells (ATCC, TIB-202) were cultured in RPMI Medium 1640 (Life Technologies, # 11875-093) plus 10% FBS. A549 cells (ATCC, # CCL-185) were cultured in RPMI Medium 1640 plus 10% FBS and 1 × MEM Non-Essential Amino Acids Solution (Life Technologies, # 11140-050).

Neonatal foreskin fibroblasts, HFF1 and BJ were purchased from ATCC (#SCRC-1041 and #CRL-2522, respectively). All cells used were cultured at 37 °C, 5% CO₂ and either 21% (standard) or 5% oxygen in an incubator under humidified atmosphere. Somatic cells were cultured in DMEM medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS) and 1x penicillin/streptomycin until reaching 90% confluency and then split in a 1:4 ratio. The conditions for passaging human pluripotent stem cells (hPSC) were a combination of methods adapted from several published protocols^{61 (link), 62} . This was applied to the culture of the human ESC-lines H1 and H9 (WiCell Research Institute, Madison, WI, USA) and iPSCs generated from NBS and HFF1 cells. In combination with MEFs, hPSCs were usually cultivated in plates coated with 0,2% gelatin and fed with hESC medium containing KO-DMEM supplemented with 20% knockout serum replacement, non-essential amino acids, L-glutamine, penicillin/streptomycin, sodium pyruvate, 0.1 mM beta-mercaptoethanol and 4 ng/ml FGF-2, which was replaced every second day.

RMS cell lines RH30, RD, RMS-YM, RH4, JR-1, RMS-01, RH41, HFF-1 have been described previously [17 (link)]. Cell lines were cultured in Dulbecco’s modified Eagle’s medium (Thermo Fisher Scientific, Waltham, MA, USA) (RD, RH30, RMS-01, RH4) or Roswell Park Memorial Institute 1640 (RPMI) medium (Thermo Fisher Scientific, Waltham, MA, USA)(RH41, RMS-YM) supplemented with 10% fetal calf serum (Gibco, Life Technologies Ltd., Thermo Fisher Scientific, Waltham, MA, USA), 2 mM L-glutamine (Thermo Fisher Scientific, Waltham, MA, USA) and 1% penicillin/streptomycin (Thermo Fisher Scientific, Waltham, MA, USA). HFF-1 cells were purchased from ATCC and cultured in Dulbecco’s modified Eagle’s medium supplemented with 15% fetal calf serum, 2 mM L-glutamine and 1% penicillin/streptomycin. Cells were maintained at 37 °C at 5% CO₂.
Cell lines were authenticated using the Geneprint 10 kit according to manufacturer’s instructions (Promega, Madison, WI, USA) and subsequent fragment length analysis was carried out by Eurofins Genomics (Ebersberg, Germany). The resulting short tandem repeat (STR) typing results were compared with public databases.

Compounds were evaluated for their anticancer activity using three cultured cell lines: SNB-19 (human glioblastoma, DSMZ – German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany), C-32 (human amelanotic melanoma, ATCC-American Type Culture Collection, Manassas, VA), MDA-MB-231 (human adenocarcinoma mammary gland, ATCC, Manassas, VA) and HFF-1 (human fibroblast cell line, ATCC, Manassas, VA). The cultured cells were kept at 37 °C and 5% CO₂. The cells were seeded (1 × 10⁴ cells/well/100 µl DMEM supplemented with 10% FCS and streptomycin and penicillin) using 96-well plates (Corning). The cells were counted in a haemocytometer (Burker’s chamber) using a phase contrast Olympus IX50 microscope equipped with Sony SSC-DC58 AP camera and Olympus DP10 digital camera.