Powerplex fusion system

The NIH-OVCAR3 cell line (ATCC HTB-161) was cultured in RPMI supplemented with 10% FBS and incubated at 37 °C in humidified atmosphere containing 5% CO₂. The growth conditions of additional 12 ovarian cancer cell lines used for comparison, including UWB1.289 and UWB1.289 + BRCA1 cell lines used as positive and negative controls for HRR assay, are listed in Table S4. M059J, DNA-PKcs-deficient human glioblastoma were cultured in DMEM supplemented with 10% FBS and incubated at 37 °C [44 (link)]. M059J-Fus-1 (M059J transfected with a portion of chromosome 8 carrying the DNA-PKcs gene) were cultured in full media supplemented with 400 μg/mL G418 [45 (link)]. Cell lines were authenticated by Short Tandem Repeat (STR) profiling using the PowerPlex Fusion System (Promega, Southampton, UK). Cell lines were mycoplasma tested every three months.

Jurkat (DSMZ, ACC 282) and DND41 (DSMZ, ACC 525) cells were cultured in RPMI 1640 GlutaMAX supplement (Thermo Fisher, 61870044) with 10% FBS. Cells were maintained in suspension culture at a density between 0.3 × 10⁶ and 1 × 10⁶. Cells were split at a ratio of 1:2 to 1:4 every 2–4 d and were frozen in 70% RPMI medium, 20% FBS and 10% dimethylsulfoxide. PEER (ACC6, DSMZ), PER-117 (a gift from U. Kees, Perth), MOLT-16 (ACC29, DSMZ), RPMI-8402 (ACC290, DSMZ), LOUCY (ACC394, DSMZ), TALL-1 (ACC521, DSMZ), ALL-SIL (ACC511, DSMZ), NALM-6 (ACC128, DSMZ), NALM-16 (ACC680, DSMZ), MHH-CALL-2 (ACC341, DSMZ), MHH-CALL-4 (ACC337, DSMZ) and MUTZ5 (ACC490, DSMZ) were maintained in RPMI 1640 medium containing 10% or 20% FBS (HyClone), penicillin/streptomycin (100 U ml⁻¹) and glutamine (100 µM) at 37 °C, 5% CO₂. Cell identity was confirmed by short tandem repeat profiling using a PowerPlex Fusion System (Promega). All cell lines were confirmed as free from Mycoplasma spp. using the Universal Mycoplasma Detection kit (American Type Culture Collection).

A polymerase chain reaction was performed using the PowerPlex^® Fusion System (Promega, Madison, WI, USA) according to [31 (link)] following the instruction manual. This system co-amplifies 23 STR loci (D3S1358, D1S1656, D2S441, D10S1248, D13S317, Penta E, D16S539, D18S51, D2S1338, CSF1PO, Penta D, TH01, vWA, D21S11, D7S820, D5S818, TPOX, DYS391, D8S1179, D12S391, D19S433, FGA, D22S1045) and the Amelogenin locus for sex determination [32 (link)]. PCR amplification was carried out using the Applied Biosystems Veriti™ Thermal Cycler (Thermofisher Scientific, Waltham, MA, USA). The PCR products (1 µL) were separated by capillary electrophoresis in an ABI 3500 Genetic Analyzer (Thermo Fisher Scientific Company, Carlsbad, CA, USA) with reference to the BTO size standard (Qiagen, Manchester, UK) in a total of 12 µL master mix consisting of the BTO size standard and Hi-Di formamide (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The analytical threshold for the PowerPlex^® Fusion analysis in GeneMapper ID-X was set at 175 RFU. However, the allele call for some peaks was cleaned manually.

All the cell lines were confirmed as Mycoplasma spp. free using the Universal Mycoplasma Detection Kit (American Type Culture Collection, Manassas, VA). Cell lines were maintained in RPMI 1640 medium supplemented with 10% FBS (Hyclone), Antibiotic (1X, Gibco), and GlutaMAX (1X, Gibco) and incubated in a 37°C humidity-controlled incubator with 5% CO₂. Each cell line identity was confirmed by STR profiling using PowerPlex Fusion System (Promega) and confirmed using ATCC or DSMZ STR database.
HEK293T cells (ATCC, Manassas, VA) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Lonza) supplemented with 10% fetal bovine serum (FBS) (Cytiva), and 100 U/mL penicillin, 100 μg/mL streptomycin, 2 mM glutamine (P/S/G) (Thermo Fisher Scientific). 697, NALM6, and REH were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% FBS and P/S/G. SUP-B15 and MHH-CALL-2 were cultured in RPMI 1640 medium supplemented with 20% FBS and P/S/G. For either coculture with human BM-MSC or mono-culture for experiments, all leukemic cell lines were cultured in α-modified Minimum Essential Medium (α-MEM) (Sigma-Aldrich) supplemented with 10% FBS, and P/S/G. All cells were incubated in a humidified incubator at 37°C with 5% CO₂.

Molecular fingerprinting for PDOXs and cell lines was performed with Promega PowerPlex® 16 HS or PowerPlex Fusion® System (Promega Corporation, Madison, WI).

Genomic DNA was purified from whole blood using the HiGene Genomic DNA Prep Kit (Biofact, Daejeon, Korea). Paternity was confirmed for all the examined families by PCR amplification of STR markers using the PowerPlex Fusion System (Promega, Wisconsin-Madison, USA) followed by the resolution of the PCR products on a SeqStudio genetic analyzer (Life Technologies-Thermo Fisher Scientific, Foster City, CA, USA).