Luciferin reagent

For overexpression studies HEK293T cells were seeded to a 24 well plate in SDM and 24 h later transfected with 150 ng pARE3-luc, 50 ng pCMV-β-gal, 10 ng pFlag-His-AR and varying combinations of pFlag-Usp12, pFlag-Uaf-1 and pHA-Flag-WDR20 at 10 ng each. All reactions were balanced with pCMV empty vector. Cells were treated with 10 nM DHT 24 h later and incubated for a final 24 h prior to lysis in 1x Reporter Lysis Buffer (Promega). Lysis solution was mixed with Luciferin reagent (Promega) according to manufacturer's instructions and luciferase counts determined using the FLUOstar Omega microplate reader (BMG LABTECH). Results were normalised to β-galactosidase activity.
For knockdown studies LNCaP-7b7 or LNCaP-AI PSA-Luc cells were subject to 96 h siRNA silencing as indicated. LNCaP-7b7 cells were grown in SDM for 72 h followed by 24 h stimulation with 10 nM DHT. LNCaP-AI PSA-Luc cells were cultured in SDM for 96 h. Luciferase activity was determined as above and results were normalised to cell density determined by live cell imaging with IncuCyte ZOOM (Essen Bioscience) immediately prior to lysis.

BRITER cells were plated in a 96-well microplate at a concentration of 20,000 cells/well and allowed to attach for 18 h. Growth media was then replaced with serum-free Dulbecco’s modified essential media (Corning, 10-017-CV) and cells were starved for 5 h. For excitation assays, cells were then treated with exogenous BMP protein serially diluted (from 500 nM to 0.049 nM) in DMEM. For inhibition assays, inhibitory proteins were diluted (from 1 μM to 0.1 nM) in DMEM with a constant amount of ligand (1 nM BMP2, 1 nM BMP2/GDF5, 1 nM BMP4/GDF5, 5 nM BMP4, 5 nM GDF5). After 3 h, cells were lysed, added to luciferin reagent (Promega, E1501), and luciferase activity was determined by measuring luminescence over 10 s with a Biotek Synergy H1 plate reader using Gen5 software. Data were normalized to untreated controls, plotted using GraphPad Prism, and analyzed by non-linear regression. All experiments were performed in triplicate. Reported values are an average of N=3 separate experiments.

Cells were transfected with PSA-luciferase reporter plasmid with the renilla reporter as a control for transfection efficiency. Luciferase activities were determined using the luciferin reagent (Promega, Madison, WI) according to the manufacturer's protocol. Transfection efficiency was normalized by renilla luciferase activity.

The in vivo metastasis models were established with 6–8-week-old immunodeficient BALB/c athymic female nude mice (Orient Bio, Seongnam, Korea). All experiments were conducted following the guidelines and protocols approved by the Institutional Animal Care and Use Committee of the Lee Gil Ya Cancer and Diabetes Institute, Gachon University (Incheon, Korea). The mice were randomly separated into five groups. MDA-MB-231 cells with DNAJB9-OE, DNAJB9-OE–FBXO45-KD, and empty vector control were harvested, and 1 × 10⁶ cells were intravenously injected into the nude mice (5 animals per group). Metastasis incidence was measured and monitored using the IVIS Spectrum in vivo imaging system (Caliper Life Sciences, Hopkinton, MA) weekly by intraperitoneally injecting 150 mg/ml luciferin reagent (Promega, Madison, WI)^{25 (link)}. The intensity of the metastasized tumors was measured using Living Image (V.3.1.0; Caliper Life Sciences). After 10 weeks, the mice were euthanized, their lung tissues were removed and fixed in 10% formalin, and lung metastases were evaluated under a microscope after hematoxylin and eosin staining. Tumor areas were measured using ImageJ (National Institutes of Health, Bethesda, MD). When mice showed severe weight loss or behavior abnormalities, they were euthanized.