br The progression of CRPC PC and Du
The progression of CRPC PC3 and Du145 cells to a docetaxel re-sistant phenotype promoted by the overexpression of PTOV1 is asso-ciated to increased expression of genes involved in resistance to doc-etaxel and self-renewal (e.g. ABCB1, CCNG2 and ALDH1A1) , suggesting that through the expression of these genes PTOV1 conferred cells a higher resistance to chemotherapy and higher plasticity [8,23–26].
Here, we studied whether PTOV1 may directly bind and activate the promoter regions of these genes in LNCaP androgen-dependent prostate cancer cells. We report the identification and localization of a novel DNA-binding motif in PTOV1 that allows the protein to directly and specifically bind to ALDH1A1 and CCNG2 promoters and it is required for their full activation.
2. Materials and methods
LNCaP androgen dependent prostate cancer cells were obtained from the American Type Culture Collection (Rockville, MD). Cells were periodically confirmed free of contaminated cell lines by in house au-thentication of cell cultures by STR-fingerprints comparing these with those published by ATCC. LNCaP cells were maintained in RPMI 1640 medium (Life Technologies, Inc., Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum (Life Technologies) at 37 °C in an atmosphere of 5% CO2. Antibodies to PTOV1 antibody were pro-duced and purified as previously described [15,27]. Wnt3a conditioned medium was kindly provided by D. Arango (Vall d’Hebron Institute of Research, Barcelona).
2.2. Plasmid and peptides
The lentiviral HAPTOV1-ires-GFP and GFP-PTOV1 vectors were previously described . Short-hairpin shRNA sequences 1397 and 1439 (Sigma-Aldrich, St. Louis, MO) targeting the human PTOV1 mRNA were as described  and are shown in Supplementary Table 3. PTOV1 mutant at Epirubicin HCl 98–100 (the sequence KRRP was changed to EGGP) was obtained with the plasmid GFP-PTOV1 using the QuikChange™ Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer's instructions. The primers used for mutagenesis were: 5′-GAGTGGCAGGAGGAGGGCGGACCCTAGTCT GAC-3’ (Forward) and 5′-GTCAGAGTAGGGTCCGCCCTCCTCCTGCCA Cancer Letters 452 (2019) 158–167
CTC-3’ (Reverse). The eAT-hook-wild type and eAT-hook-mutant pep-tides were purchased from PepMic (Suzhou, China). The A-domain peptide was described previously .
Total RNA was extracted with the RNeasy mini kit (QIAGEN, Hilden, Germany), reverse transcribed with the Mooney Murine Leukemia Virus Reverse Transcription (M-MLV-RT) kit (Promega, Madison, USA) and real-time qPCR performed with the Universal Probe Library (Roche, Basilea, Switzerland) on a LightCycler 480 RealTime PCR instrument (Roche). Primers are shown in Supplementary Table 1. The Ct method was applied to estimate relative transcript levels. TBP, IPO8, or HMBS were used as endogenous control genes. Values are presented as mean ± SD.
2.4. Chromatin immunoprecipitation (ChIP)
Chromatin was immunoprecipitated using EZ-chip Chromatin Immuno Precipitation kit (Millipore, Burlington, USA) according to the manufacturer, as previously described .
Briefly, after a mild formaldehyde crosslinking step, cells were so-nicated, lysates incubated with primary antibodies and precipitated with protein A/G-Sepharose. Crosslinking of DNA-protein complexes was reversed, DNA purified and used as a template for PCR reactions. Primers used for PCR in ChIP experiments are described in Supplementary Table 2.
2.5. Electrophoretic mobility shift assay (EMSA)
For binding assays, we used the following double-stranded DNAs corresponding to regions of: ALDH1A1 promoter (from −395- to −363), CCNG2 promoter (from −269 - to −239) and HES1 gene (from +45 to +69). The dsDNA probes were formed by mixing 20 μg of each single-stranded oligodeoxynucleotide in a 150 mM NaCl solution. After incubation at 90 °C for 5 min, solutions were allowed to cool down slowly to room temperature. The duplexes were purified in a non-denaturing 20% polyacrylamide gel electrophoresis and DNA con-centration was determined by measuring its absorbance (260 nm) at 25 °C in a Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Barcelona, Spain). Duplexes were 5′-end-labeled with [γ-32P]-ATP (Perkin Elmer, Madrid, Spain) by T4 polynucleotide kinase (New England BioLabs) in a 10 μL reaction mixture, according to the manufacturer's protocol. After incubation at 37 °C for 1 h, 90 μL of Tris-EDTA buﬀer (1 mM EDTA and 10 mM Tris, pH 8.0; Sigma-Aldrich) were added to the reaction mixture, which was subsequently filtered through a Sephadex G-25 (Sigma-Aldrich) spin-column to eliminate the unin-corporated [γ-32P]-ATP. Radio-labeled probes (100.000 cpm, [γ-32P]-ATP) were placed in ice for 30 min with 10 μg of PTOV1 domains or 0.3 μg of peptides using either 5 μg or 0.15 μg poly (dI:dC), respectively, as unspecific competitor, in the presence of the binding buﬀer (5% Glycerol, 0.5 mM DTT, 4 mM MgCl2, 36 mM KCl, 0.5 mM EDTA, 25 mM Tris-HCl pH 8.0; all reagents were purchased from Sigma-Aldrich). The products of the binding reactions were electrophoretically resolved in 5% polyacrylamide and 5% glycerol native gels at a fixed voltage of 220 V and 4 °C. Gels were dried at 80 °C and visualized on a Storm 840 PhosphorImager (Molecular Dynamics, GE Healthcare Life Sciences, Barcelona, Spain). ImageQuant software v5.2 was used to visualize the results.