Tet on system

7k27OHC, 7β27OHC, 7α27OHC, and 7β27OHC-d6 were obtained from Avanti Polar Lipids, Inc. (Alabaster, AL), [1,2-³H]cortisone, [1,2,6,7-³H]cortisol, and [1,2,6,7-³H] corticosterone from American Radiolabeled Chemicals (St. Louis, MO), and all other chemicals from Sigma-Aldrich (Buchs, Switzerland) of the highest grade available. Cell culture media were purchased from Sigma-Aldrich and Invitrogen (Carlsbad, CA) and FBS approved for use with the Tet-on system from Clontech (Mountain View, CA). UHPLC-grade purity methanol, acetonitrile, and formic acid were from Biosolve (Dieuze, France). 5H-1,2,4-triazolo(4,3-a)azepine,6,7,8,9-tetrahydro-3-tricyclo(3-3-1-13-7)dec-1-yl [T0504; also known as Merck-544 (25 (link))] was purchased from Enamine (Kiev, Ukraine).

Clontech’s Tet-On system was used to generate a stable E4-ORF3-inducible cell line. A PCR-generated amplicon consisting of the Ad2 E4-ORF3 coding region was cloned into the BamHI and EcoRI restriction enzyme sites of the pUHD10-3 vector, fused at the N-terminus with an HA epitope and placing it under the control of a doxycycline (dox)-inducible promoter element (pTet-HA-E4-ORF3). The resulting plasmid construct was cotransfected into U2OS-Tet-On osteosarcoma cells with pTK-Hyg (Clontech) using Fugene 6. Two days post transfection, cells were treated with 200 μg/mL G418 and 100 μg/mL Hygromycin B to select for cells with pTet-HA-E4-ORF3. Individual, drug-resistant colonies were isolated, amplified, and treated with 1 μg/mL dox for 48 h to screen for HA-E4-ORF3 expression. One cell subclone that exhibited no E4-ORF3 expression minus the addition of dox and levels of E4-ORF3 equivalent to that observed following Ad5 infection was chosen for subsequent use (termed Tet-E4-ORF3 cells).

Tet-inducible cell lines were established by inserting p14^ARF-GFP-IRES-Luciferase cassettes into the Tet-on system (Clontech). First, the Tet receptor (Tet-R) expressing plasmids were introduced into LNCaP or C4-2B cell lines and selected by zeocin antibiotics. Secondly, stable cell lines of Tet-R-LNCaP or Tet-R-C4-2B were transfected with plasmids containing p14^ARF-GFP-IRES-Luciferase and selected by Puromycin. For IGF stimulation, 22Rv1 cells were treated with IGF at a concentration of 100 μg/ml for 6 hrs. For migration assay, cells were grown until 70–80% confluence followed by serum starvation for 40 hrs. Cells were seeded at a density of 5 × 10⁴/well for PC3 cells or 7.5 × 10⁴/well for LNCaP cells in serum-free medium into the upper chamber with 8 μm polyethylene terephalate membrane filters (Falcon cell culture insert, Becton-Dickinson). Cells were allowed to migrate for 24 hrs for PC3 cells or 48 hrs for LNCaP cells in a humidified chamber at 37°C with 5% CO₂. Non-migrant cells on the upper side of the filters were removed using cotton swab. Filters containing cells were fixed with 4% formaldehyde for 15 min, and cells in the lower filter were stained with 0.5% crystal violet and counted from three microscopy fields.