Rwpe 1

The human PCa cell lines 22RV1, LNCaP, DU145, PC-3, VCaP, and normal.
prostate epithelial cells RWPE-1 were purchased from the Shanghai Chinese Academy of Sciences Cell Bank (China). RWPE-1 cells were cultured in Keratinocyte-SFM (1×) (Invitrogen, USA). PC-3, 22Rv1, and LNCaP cells were grown in RPMI-1640 medium (Life Technologies, USA) supplemented with penicillin G (100 U ml^− 1), streptomycin (100 mg ml^− 1) and 10% fetal bovine serum (FBS, Life Technologies, USA). The C4-2B cell line was purchased from the MD Anderson Cancer Center and cultured in T-medium (Invitrogen) supplemented with 10% FBS. DU145 and VCaP cells were grown in Dulbecco’s Modified Eagle’s Medium (Invitrogen, USA) supplemented with 10% FBS [7 (link)].

Primary HMEC and human prostate epithelial cells (PHECs) were ordered from from Clonetics BioWhittaker (Walkersville, MD, USA). MCF-10F, RWPE1, U2OS, and 293T cells were ordered from ATCC. MGC-803 cells were obtained from Cell Resource Center, Chinese Academy of Medical Sciences, China. The HMEC/HMEC-hTERT and PHEC/PHEC-hTERT cells were maintained in serum-free mammary (Invitrogen) and prostate (PrEBM, Clonetics BioWhittaker) basal medium supplemented with growth factors, respectively. RWPE1 cells were cultured in keratinocyte serum-free medium (Invitrogen). U2OS and 293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS). MCF-10F cells were cultured in DMEM/F12 (1:1) media containing 5% horse serum (Invitrogen), 10 μg/mL insulin, 20 ng/mL epidermal growth factor, and 500 ng/mL hydrocortisone. MGC-803 cells were cultured in RPMI-1640 supplemented with 10% FBS. All cultures were kept at 37 °C with 5% CO₂ atmosphere. Mycoplasma was tested as negative using Mycoplasma Detection Kit (Biotool, USA).

Human breast carcinoma cell line BT474 [31 (link)] was cultured in RPMI 1640 with 10% FBS, sodium pyruvate (10mM), N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (10mM) and antibiotics. Androgen-independent human PC-3 prostate cancer cells [32 (link)] were obtained from ATCC (Rockville, MD). Cells were cultured in RPMI 1640 (Bio-Whittaker, Walkersville, MD) supplemented with 10% FBS (Hyclone, Logan, UT), penicillin (100U/ml), streptomycin (100ug/ml), sodium pyruvate (1 mM) and non-essential amino acids (0.1 mM) under conditions indicated in the figure legends. Normal prostate epithelium cells RWPE-1 [33 (link)] were obtained from ATCC (Rockville, MD). RWPE-1 cells were maintained in Keratinocyte-Serum Free medium (Invitrogen, Carlsbad, CA) supplemented with 5 ng/ml of human recombinant EGF and 0.05 mg/ml of bovine pituitary extract. Ovarian cancer cell lines [34 (link)] were a kind gift from the Dennis Connolly lab. The NKE cells [35 (link)] were obtained from ATCC (Rockville, MD). The PNX cell line was a kind gift from Dr. Igor Astsaturov, MD, PhD (Fox Chase Cancer Center, Philadelphia, PA). Tumor cells were isolated from tumor tissue specimen obtained with written informed consent and Fox Chase Cancer Center Institutional Review Board approval (IRB approved protocol #12-822) from a patient undergoing tumor resection at the Fox Chase Cancer Center.

Normal human prostate epithelial cell line, RWPE-1, was obtained from the American Type Culture Collection (ATCC, Manassas, Virginia). For CRISPR knockout, cells were transfected using Lipofectamine as per the manufacturer’s protocol with commercially available Sigma (St. Louis, Missouri) CRISPR plasmid expressing gRNA targeting the first coding exon of TET2, as well as Cas9 fused to GFP via a 2A linker peptide (gRNA target ID: HS0000114238; sequence: TTAGTAGCCTGACTGTTAA with TGG protospacer-associated motif). Forty-eight hours post-transfection, GFP-FACS was used to perform single-cell sorting of successfully transfected cells onto 96-well plates. Post-expansion, populations were assayed via Sanger sequencing (The Centre for Applied Genomics, Toronto) for the presence of indels. Off-target analysis was performed as described by Mali et al. [27 (link)]. RWPE-1 and knockout cells were cultured with keratinocyte serum-free medium (K-SFM) (Invitrogen, catalog #17005042), supplemented with 0.05 mg/mL bovine pituitary extract (BPE) and 5 ng/mL human recombinant epidermal growth factor (EGF). All cells were cultured as a monolayer and maintained in a humidified incubator at 37 °C with 5% CO₂.