Geneprint 10

STR analysis was performed at the SickKids Center for Applied Genomics Facility using GenePrint10 (Promega).

Patient-derived xenografts (PDX) were established by the Melbourne Urological Research Alliance (MURAL). Written informed consent was obtained from all patients following Human Research Ethics Committee (Institutional Review Board) approvals from the Peter MacCallum Cancer Centre (11/102), Cabrini Health (03-14-04-08), and Monash University (1636, 12287). The studies were conducted in accordance with the National Statement on Ethical Conduct in Human Research produced by the National Health and Medical Research Council of Australia. All animal handling and procedures were approved by the Monash University Standing Committee of Ethics in Animal Experimentation (MARP 2012/158 and MARP/2014/085). CRPC PDXs 27.2 and 167.2 (19 (link)) were grown in an ex vivo culture system as described previously (20 (link)). Tumor fragments were treated for 48 hours with 10 or 100 nmol/L MeT, DHT or vehicle control. IHC of Ki67 and CC3 was performed as described previously (20 (link)). PDXs were routinely authenticated using short tandem repeat profiling (GenePrint 10, Promega) at the Australian Genome Research Facility.

Previously published [20 (link)] KAI3 NK cells engineered to express STAT3 wildtype (STAT3^WT) or activating mutations of STAT3 (STAT3^G618R and STAT3^Y640F) were used to validate the functional effects of STAT3 mutations. Cells were maintained in RPMI 1640 Medium containing 10% FBS, 1% penicillin/streptomycin, 2 mM L-glutamine, and 100 IU/ml recombinant human Interleukin-2. Mycoplasma test was performed using PCR Mycoplasma Test kit (Cat. PK-CA91-1096, PromoCell, Heidelberg, Germany). Cells were authenticated using GenePrint10 (Promega, Madison, WA, USA). The DSMZ-STR, ATCC-STR, JCRB-STR, and ICLC-STR databases were used to compare the results.

Unless stated otherwise, all cell lines were verified by genetic profiling of polymorphic short tandem repeat (STR) loci as per ATCC standards. We used AmpFISTR test or GenePrint10 test (Promega, Madison, WI) and analysed all the data using GeneMapper v4.0 software. LNCaP, C4-2, PC3, PNT1a, and DUCaP cells were obtained from commercial suppliers and grown in RPMI cell culture medium containing 10% FBS and 1% Penicillin/Streptomycin in a humidified incubator at 37 °C with 5% CO₂. We generated and cultured C4-2-NT control (expressing non-targeting RNA; siNT) and C4-2-shAR cells as described earlier^{19 (link)}. LNCaP-LN3 cells have been described previously^{26 (link)}.

Xenograft tumour tissue were cut into small pieces in RPMI-1640 medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS) (Gibco), 100 U penicillin G, and 100 µg/mL streptomycin (Gibco). The small tissue pieces were treated with collagenase 2 (Worthington) at 37 °C for 5 min and then dissociated mechanically by passage through an 18-gauge (1.02 mm) needle. Cell suspensions were filtered with a 40-μm nylon mesh (BD Falcon) and seeded in a 10-cm culture plate at 37 °C in a humidified atmosphere of 5% CO₂. Weakly adherent cells were maintained for >12 months in culture and passaged >50 times. Cells continuously expressed the CIC-DUX4 transcript throughout establishment, as determined by RT-PCR. The absence of contaminated mycoplasma was examined with e-Myco Mycoplasma PCR Detection Kit (Intron biotechnology). Cell lines were authenticated with GenePrint 10 (Promega).

SK-MEL 05 and SK-MEL-147 human melanoma cell lines (both wild type p53^{31 (link),32 (link)}) were authenticated by analysis of short tandem repeats, GenePrint 10 (Promega Internal, Standard-ILS 600, performed by the Rede Premium Core Facility, FMUSP) and tested negative for mycoplasma by a PCR assay using conditioned medium as template and amplification using the following oligonucleotides:

Myco F: 5′-GGG AGC AAA CAC GAT TAG ATA CCC T-3′.

Myco R: 5′-TGC ATT ATC TGT CAC TCT GTT AAC CTC-3′.

These cell lines as well as HEK293 were cultivated in DMEM with 10% fetal calf serum, supplemented with antibiotic–antimycotic (Thermo Fisher Scientific, Waltham, MA, USA) and maintained at 37 °C and 5% CO₂ atmosphere.