Mda t41

The study included five TC-derived cell lines (MDA-T32, MDA-T41, U-hth-74, U-hth-104, and SW1736). ATC-derived cell lines U-hth-74, U-hth-104 and SW1736 were obtained from Dr. N-E Heldin and cytogenetically characterized [30 (link)]. Short tandem repeats (STR) genotyping for ATC-derived cell lines U-hth-74, U-hth-104 and SW1736 was recently performed and matched to previously published genotypes [9 (link)], while the PTC-derived cell lines MDA-T32 and MDA-T41 were purchased from ATCC in 2018. Cells were cultured in RPMI-1640 medium (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA), 100 U/mL penicillin, 100 μg/mL streptomycin, and 4 mM L-glutamine.

BCPAP, TPC1 and 8505c were obtained from Dr.Santoro, University of Naples, Nthy-ori 3–1 from Dr.Greco, Foundation IRCCS-INT, Milan. 8305c were purchased from Merck KGaA (Darmstadt, Germany). MDA-T41 were purchased from ATCC (Manassas, Virginia, USA). TPC1 and BCPAP were cultured in DMEM while the other cell lines in RPMI at 37 °C/5% CO2 in medium added with 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA) and 1% penicillin – streptomycin (Euroclone, Milan, Italy). MDA-T41 medium was added with non-essential amino acids (Thermo Fisher Scientific, Waltham, MA, USA). All cell lines were routinely tested for Mycoplasma contamination using Lonza™ Mycoalert™ Mycoplasma Detection Kit (Thermo Fisher Scientific, Waltham, MA, USA). Authentication by SNP profiling at Multiplexion GmbH (Heidelberg, Germany) was performed in December 2018 for TPC1, BCPAP and 8505c. Authentication by PCR-single-locus-technology at Eurofins Medigenomix Forensik GmbH (Ebersberg, Germany) for Nthy-ori 3–1 was performed in March 2019.

The study included three TC-derived cell lines U-hth-74, U-hth-104, and MDA-T41. Cells were cultured in RPMI-1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific), 100 U/mL penicillin, 100 μg/mL streptomycin, and 4 mM L-glutamine. The ATC-derived U-hth-74 and U-hth-104 cell lines carry a C228T TERT promoter mutation as verified by Sanger sequencing [19 (link)], while the PTC-derived MDA-T41 carries a wild-type (WT) TERT promoter [26 (link)]. For DNA methylation inhibition, U-hth-74 and U-hth-104 cells seeded in 6-well plates were treated with 10 μM of 5-Azacytidine (5-Aza, Sigma-Aldrich, Darmstadt, Germany) for 72 h. During the 72 h of culturing, medium was changed every 24 h. Control cells were incubated with the same volume of DMSO. ATC-derived cell lines U-hth-74 and U-hth-104 were obtained from Dr. N-E Heldin and short tandem repeats (STR) genotyping was previously performed and matched to previously published genotypes [19 (link),27 (link)]. MDA-T41 was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). All cell experiments were repeated three times unless stated otherwise.