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  • Navitoclax br Our results demonstrated that


    Our results demonstrated that ADT induces ZBTB46 expression through the downregulation of the androgen-responsive SAM pointed domain containing ETS transcription factor (SPDEF), leading to in-creased expression of PTGS1 and contributing to NE differentiation of prostate cancer cells. The addition of PTGS1 inhibitor treatment can restore enzalutamide (MDV3100) sensitivity and reduce tumor growth, whereas overexpression of ZBTB46 disrupts the tumor-suppressive ef-fect of this combination treatment and induces PTGS1- and NEPC-as-sociated genes. Our findings provide a novel link between NE differ-entiation and the expressions of inflammatory response genes as well as new biomarkers and therapeutic targets for NEPC.
    2. Materials and methods
    2.1. Cell lines and cell culture
    The AR-positive C4-2B and LNCaP-AR (parental LNCaP over-expressing wild-type AR [32]) and AR-negative RasB1 (an aggressive cell line expressing a constitutively active Ras in the DU145 Navitoclax and isolated from a bone metastasis [27,33–40]) cell lines were obtained from Dr. Kathleen Kelly (NCI/NIH, MD, USA) and maintained as pre-viously described. The PC3, LNCaP, C4-2, and 22Rv1 cell lines were from ATCC (VA, USA), and they were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS). The NE-like NE-1-8 (a subclone cell from the LNCaP cell line that is treated with long-term ADT [41]) and TRAMP-C1 (a more NE mouse prostate cancer model [42–46]) cell lines were purchased from ATCC and separately cultured in RPMI 1640 medium supplemented with 5% charcoal-stripped serum (CSS) and DMEM medium supplemented with 10% FBS, 0.005 mg/ml 
    bovine insulin (Sigma-Aldrich), and 10 nM dehydroisoandrosterone (Sigma-Aldrich). The AR antagonist, enzalutamide (MDV3100) (Selleck), and the PTGS inhibitor, NS-398 (Sigma-Aldrich), were used separately to treat the cells at 10 μM and 1.77 or 75 μM for 24 h in 10% FBS-containing medium. Dihydrotestosterone (DHT) (Sigma-Aldrich) was used to treat cells at 10 nM for 24 h in 10% CSS-containing medium.
    2.2. Chromatin immunoprecipitation (ChIP) assay
    ChIP assays were performed using an EZ magna ChIP A kit (Millipore) with a modified protocol. C4-2B and LNCaP-AR cells were treated with DHT (Sigma-Aldrich) at 10 nM for 4 h in 10% CSS-con-taining medium or MDV3100 (Selleck) at 10 μM for 4 h in 10% FBS-containing medium, as described in Supplementary Methods.
    2.3. Promoter reporter assay
    For the promoter reporter assays, LNCaP, RasB1, or C4-2B cells in 12-well plates (5 × 104 cells/well) were transiently transfected with 1 μg of the PTGS1- or ZBTB46-green fluorescent protein (GFP) reporter containing ZBTB46- or SPDEF-binding sites. The cells were treated with DHT (Sigma-Aldrich) and MDV3100 (Selleck) or small interfering (si) RNA and DNA (empty vector (EV), ZBTB46- or SPDEF-expressing vector) by transfection, as described in Supplementary Methods.
    2.4. Proliferation assay
    The cells were stably transfected with a gene encoding an SPDEF or ZBTB46 expression vector or a ZBTB46 or SPDEF short hairpin (sh)RNA vector and seeded at a density of 2000 cells/well in 96-well plates. The cells were treated with MDV3100 at 10 μM and NS-398 at 1.77 or 75 μM for 24 h in 10% FBS-containing medium, as described in Supplementary Methods.
    2.5. Colony-formation assay
    A colony-formation assay was performed using a starting number of 1000 cells/well seeded in 0.3% agarose (Affymetrix) on top of a 0.6% agarose layer in 12-well plates. Single-cell suspensions of ZBTB46 complementary (c)DNA-transfected 22Rv1 and C4-2B cells, SPDEF/ ZBTB46 cDNA-transfected RasB1 cells, or ZBTB46 shRNA vector-transfected PC3 and RasB1 cells were performed as described in Supplementary Methods.