Identifiler kit

Human thyroid cancer cell lines C643, SW1736, BCPAP, and Cal62 were grown in RPMI (Invitrogen, Carlsbad, CA) supplemented with 5% FBS (HyClone Laboratories, Logan, UT), and the A375 cell line was grown in DMEM and supplemented with 10% FBS. All lines were maintained at 37°C in 5% CO₂. All cell lines were validated using short tandem repeat profiling using the Applied Biosystems Identifiler kit (#4322288) in the Barbara Davis Center BioResources Core Facility, Molecular Biology Unit, at the University of Colorado, as previously described (27 (link)). The SW1736 and C643 cells were generously provided by Dr. K. Ain (University of Kentucky, Lexington, KY), with permission from Dr. N.E. Heldin (University Hospital, Uppsala, Sweden). The BCPAP and Cal62 cells were generously provided by Dr. M. Santoro (Medical School, University “Federico II” of Naples, Naples, Italy). All cell lines were routinely monitored for Mycoplasma contamination using the Lonza Mycoalert system (Lonza Walkersville, Inc., Walkersville, MD), according to the manufacturer's directions.

Division of Cancer Treatment and Diagnostics of the National Cancer Institute (DCTD/NCI) collected a panel of 63 human adult and pediatric sarcoma cell lines. Cells were purchased from ATCC (Manasses VA), or obtained from Dr. Samuel Singer (Memorial Sloan Kettering Cancer Center, NY, NY), the Children’s Oncology Group (COG; Dr. Patrick Reynolds, Texas Tech University Health Sciences Center, Lubbock, TX) and Dr. Peter Houghton (Nationwide Children’s Hospital, OHSU). The atypical synovial sarcoma cell line, SW982 expresses SSX gene, but not SYT-SSX or HLA-A24 (Supplemental Figure 1). The ASPS-1 aveolar soft part sarcoma line was developed at NCI (20 (link)). The sarcoma lines were stored frozen at 10⁶ cells per ml in liquid nitrogen. The sarcoma lines were authenticated using the Applied Biosystems Identifiler kit for short tandem repeat analysis (15 loci). The lines were thawed from the banked stock and samples were taken for Identifiler analysis within passages 2–5. New cells from the same frozen stock were thawed after a maximum of 20 passages, which did not exceed 5 continual months in culture. The human A549 NSCLC line purchased from ATCC was run on each plate as a screen control. The lines were maintained in the medium specified for each line supplemented with fetal bovine serum and other additives (Supplemental Table 1).

Caki-1, HK2 and A-704 cells were purchased from American Type Culture Collection (ATCC) in April 2015, and authenticated by STR DNA fingerprinting (on 30^th June [Cak1-1 and HK2] and 20^th August 2015[A-704]) at the University of Michigan DNA Sequencing Core using Identifiler+kit (Applied Biosystems, Inc) and the resulting amplicons were analyzed in ABI3720XL Genetic Analyzer. Routine mycoplasma testing was also performed using MycoAlert Plus kit (Lonza) every three weeks while in culture, following manufacturers recommendations. The cells were grown in McCoy, Keratinocyte and RPMI media supplemented with 10% FBS, penicillin, and streptomycin respectively. siRNAs were obtained from Ambion and transfected (25nM) using Lipofectamine RNAimax (Invitrogen). For transfection 0.4 million cells were plated in a 6-well plate before transfection. 48 hrs after transfection cells were trypsinized, counted and replated for proliferation assay. Remaining cells were used for RNA isolation using miRNA easy kit (Qiagen). RNA was converted to cDNA using Superscript III (Invitrogen) to perform qRT-PCR.