Pgl4.50 luc2 cmv hygro vector

PC3-M cells were transfected with the pGL4.50 [luc2/CMV/Hygro] vector (Promega) using FuGene HD transfection reagent (Promega) following the manufacturer's instructions. Individual colonies were isolated by ring cloning and 3 colonies that stably expressed the highest bioluminescent signals were identified using D-luciferin (Promega) with a Varioskan Flash Reader (Thermo Scientific). The relationship of the luminescent intensity with the number of cultured cells was assessed by an IVIS imaging system (Perkin Elmer). Cells (5×10⁵) from PC3-M- luc2 colony were suspended in 30μL PBS and orthotopically implanted into 24, 8-10 weeks old male Balb/c nude mice (Charles River, UK) by direct injection into the dorsal prostate. Mice are too small in which to distinguish zonal structuresof the prostate [48 (link)]. One-week later, tumour-bearing mice were divided into 3 groups (8 each) and subjected to the following intraperitoneal injections: 1) control with PBS; 2) dmrFABP5 (20mg/kg); 3) dmrFABP5 (20mg/kg) plus SBFI26 (1mg/kg). Injections were repeated every two days for 25 days and the metastatic foci were monitored weekly using IVIS after mice were injected subcutaneously with D-luciferin (150 mg/kg). Bioluminescent images were analysed using the Living Imagine software (Xenogen) and recorded as total photons/second (p/s) within each defined region.

PC3-M cells were transfected with the pGL4.50 [luc2/CMV/Hygro] vector (Promega) using FuGene HD transfection reagent (Promega) following the manufacturer’s instructions. Individual colonies were isolated by ring cloning and 3 colonies that stably-expressed the highest bioluminescence signals were identified using D-luciferin (Promega) with a Varioskan Flash Reader (Thermo Scientific). Association of the luminescence intensity with the number of cells was assessed by an IVIS imaging system (Perkin Elmer). Cells (5 × 10⁵) from PC3-M- luc2 colony were suspended in 30 μL PBS and orthotopically implanted into the dorsal prostate of 33 male Balb/c nude mice (Charles River, UK) (8–10 weeks old), as described previously [55]. One week later, tumour-bearing mice were divided into 2 groups (8 each) and subjected to the following intraperitoneally injections: 1) control with PBS; 2) SBFI26, 1mg/kg. Injections were repeated every two days for 25 days and the metastatic loci were monitored weekly using the IVIS after mice were subcutaneously injected with D-luciferin (150 mg/kg). Bioluminescence images was analysed using the Living Imagine software (Xenogen) and the measurement recorded was based on total photons/second (p/s) within each defined region of interest.

A metastatic subline of the human A375 IV GFP cell line was cultured in Dulbecco’s modified Eagle’s medium (DMEM, 31966-021, Gibco, Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS, 10270-106, Gibco, Life Technologies, Grand Island, NY) and 1% penicillin–streptomycin (XC-A4122/100, Biosera, Boussens, France) at 37 °C in a 5% CO₂ atmosphere and 95% relative humidity. The cells were transfected with the pGL4.50 (luc2/CMV/Hygro) Vector (E1310, Promega, Madison, WI) containing resistance to Hygromycin B to enable stable luciferase expression and selection of clones in colony formation assays (Section "Colony formation assay"). Clonal lines were generated from single-cell-derived colonies and validated for GFP and firefly luciferase expression. The AmpFLSTR Identifiler PCR Amplification Kit (Applied Biosystems, ThermoFisher Scientific, Waltham, MA) was used to confirm the origin of cell lines. Cell lines were regularly tested for mycoplasma contamination using PCR.