Cell lines
The human 293 T and PC3 cell lines were provided by the American Type Culture Collection (ATCC, Rockville, MD). The canine prostate cancer CHP-1 cell line was established from a prostate mass collected immediately following surgery of a tumour-bearing, 10-year-old, castrated male Jack Russell Terrier breed dog in our university [5]. The 293 T and the CHP-1 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Wako, Osaka, Japan) and PC3 cells were maintained in Ham’s F12 medium (Wako) supplemented with 10% foetal bovine serum (FBS), penicillin (50 IU/mL), and streptomycin (50 μg/mL) under a humidified atmosphere with 5% CO2 at 37 °C.
Mammalian two-hybrid (MTH) assay
For the MTH assay, the full-length open reading frame (ORF) of canine SGTA and REIC/Dkk-3 cDNA was cloned between the EcoRI and MluI sites of a pM GAL4 DNA-binding domain cloning plasmid (GAL4-DBD) and pVP16 transactivation domain cloning plasmid (VP16-AD) (Clontech Laboratories, Palo Alto, CA, USA), respectively. In addition, the full-length ORF of canine REIC/Dkk-3 cDNA was cloned between the XhoI and EcoRI sites of the pMACS KK HA(C) vector cloning plasmid (Miltenyi Biotec, Bergisch Gladbach, Germany). Primers were used in this study was described in Additional file 1: Table S1. Approximately 2 × 105 293 T cells per well in 24-well plates were co-transfected with 100 ng of pM, pVP16, pMACS KK HA(C), and pFR-Luc firefly luciferase reporter plasmid (Promega, Madison, WI, USA), and 0.2 ng of phRL-TK Renilla luciferase reporter plasmid (Promega). The cells were harvested 48 h after transfection, and the luciferase activity was measured using the dual-luciferase reporter assay system (Promega). The luciferase activity was normalized to the value of the Renilla luciferase activity [21].
Pull-down (PD) assay
A Halo-tagged canine SGTA was cloned into a pFN21A vector (Promega). To generate haemagglutinin (HA)-tag fusion proteins, the XhoI/EcoRI fragment of canine REIC/Dkk-3 cDNA was cloned into a pMACS KK HA(C) vector. The expression of Halo and HA-tagged constructs was induced in 293 T cells using the FuGENE® HD transfection reagent (Promega), and the transfected cells were grown for 48 h. Cells were harvested by centrifugation and washed with Phosphate-buffered Saline (PBS). The cells were lysed in Mammalian Lysis Buffer (Promega) with a Protease Inhibitor Cocktail (Promega) for 15 min, and the cellular debris was cleared by centrifugation. To the supernatant, 100 μL of HaloLink™ Resin (Promega), equilibrated with TBS including 0.05% IGEPAL CA-630 (TBS+), were added. The samples were incubated for 20 min at 25 °C with rotation. The supernatant was discarded; the resin was washed three times with TBS+ and resuspended in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer. The samples were analysed by western blot analysis using an anti-Halo antibody (1:2000) (G9281, Promega), an anti-haemagglutinin (HA) tag antibody (1:2000) (MBL-561, Medical and Biological Laboratories (MBL), Aichi, Japan) (1:2000), and a horseradish peroxidase-conjugated anti-rabbit IgG antibody (GE Healthcare, Waukesha, WI, USA). Blots were developed using the EzWestLumi plus reagent (ATTO, Tokyo, Japan).
Immunostaining
Immunocytochemical co-staining for forced-expressed Halo-tagged canine REIC/Dkk-3 and endogenous SGTA in CHP-1 cells was performed using the mouse monoclonal anti-Halo antibody (1:100) (G9211, Promega) and rabbit polyclonal anti-SGTA antibody (1:100) (sc-292,025, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Cells were plated and cultured to 30–40% confluence in Lab-Tek chambers (Nalgene, Rochester, NY, USA), and were transfected with the pFN21A vector containing Halo-tagged canine REIC/Dkk-3 by FuGENE HD (Promega). Forty-eight hours after transfection, the cells were fixed in 4% paraformaldehyde in 100 mM phosphate buffer and blocked with 5% normal goat serum in PBS. After the samples were incubated overnight at 4 °C with primary antibodies, they were incubated for 1 h at 25 °C with Alexa Fluor 488 anti-mouse and Alexa Fluor 594 anti-rabbit secondary antibodies (Invitrogen, Carlsbad, CA, USA). To stain the nuclei, the cells were incubated with Hoechst 33,342 (Dojindo Laboratories, Kumamoto, Japan) for 15 min at room temperature. Fluorescent staining was visualised and analysed under a fluorescence microscope system equipped with an analytical software program (BZ-9000, Keyence, Osaka, Japan).
Transactivation assays
For this experiment, 10% charcoal-stripped FBS was used for the cell culture. Both PC3 and CHP-1 prostate cancer cells (2 × 105/well in 24-well plates, 500 μL medium/well) were transfected with 100 ng of pEGFP C1 AR vector containing the full-length AR (Plasmid ID: 28,235, Addgene, Cambridge, MA, USA) [22], 100 ng of p159-pPr-luc vector containing a firefly luciferase reporter gene downstream of the rat probasin gene promoter (Plasmid ID: 8392, Addgene) [23], 100 ng of pFN21A-SGTA, 200 ng of pMACS KK HA(C)-REIC/Dkk-3, and 25 ng of phRL-tk Renilla luciferase reporter plasmid by using the Lipofectamine 2000 reagent (Thermo Fisher Scientific, Waltham, MA, USA). After 48 h of transfection, the cells were treated for 24 h with control vehicle (ethanol) or dihydrotestosterone (DHT) (Sigma, St Louis, MO, USA), and were assayed for luciferase activity using a Dual-Luciferase Reporter Assay System (Promega). All transfection mixes were balanced with the appropriate empty vectors in terms of the ratio of the expression vectors and total plasmids.
Immunoblotting
Total protein was extracted from the cells using Mammalian Lysis Buffer (Promega) with Protease Inhibitor Cocktail (Promega) for 15 min, and the cellular debris was cleared by centrifugation. Canine control fibroblasts were collected from normal breast tissue of an 8-year-old female Chihuahua undergoing contraceptive treatment. Western blot analysis was performed as previously described [24]. Approximately 10 μg of extracted protein were analysed with the specific primary antibodies as follows: rabbit polyclonal anti-EGFP (MBL-598, MBL), rabbit polyclonal anti-SGTA (sc-292,025), rabbit polyclonal anti-REIC/Dkk-3 (10365–1-AP, Proteintech, Chicago, IL, USA), and anti-β-actin (sc-69,879, Santa Cruz Biotechnology).
Statistical analysis
The data are shown as the mean ± SE. Student’s t-test or one-way analysis of variance (ANOVA) followed by Bonferroni test were performed to assess the significance of differences between two or more groups, respectively. Differences were considered significant with P < 0.05.