Pgl4.18 luc2p neo

A 1.5 kb fragment of the proximal IGFBP2 promoter was amplified with the following primer set: IGFBP2-For: 5′-CAGGGTACCCTGTGCCCTTGCTAACCGCCCATTTC-3′, IG FBP2-Rev: 5′-CAGGCTAGCCGGGTCCTAAGGGCCGGCTTCTCC-3′ (restriction enzyme site KpnI and NheI were underlined). The DNA fragment was inserted into the luciferase reporter vector pGL4.18 [luc2P/Neo] (Promega Corporation, Madison, Wisconsin, United States) by KpnI and 3′ NheI (IGFBP2-pGL4.18). For luciferase reporter assay, 5 × 10⁴ HEK293T cells were cultured in 12-well plates overnight. Next day, cells were transfected with 1 μg of with lentiviral empty vector or lenti-RUNX2 together with 20 ng of IGFBP2-pGL4.18 and Renilla luciferase pGL4.74 [hRluc/TK] plasmid (Promega, Madison, WI, United States) using PolyJet^TM In Vitro DNA Transfection Reagent (SignaGen Laboratories, Rockville, MD, United States). At 48 h post transfection, luciferase activity in each well was measured by Dual-Luciferase^® Reporter Assay System (Promega) and normalized to Renilla.

Plasmid pGL4‐ARR2PB‐luc was constructed by cloning ARR2PB fragment into pGL4.18 [luc2P/Neo] (#E6731, Promega). Plasmid pCMV‐hAR (#89 078, Addgene) was a gift from Elizabeth Wilson. Plasmids hAR^F876L, hAR^F876L/T877A, hAR^T877G, and hAR^W741C were constructed by site‐directed mutagenesis of pCMV‐hAR through overlap extension PCR.
PC3 cells were propagated in RPMI‐1640 (#MA0215, Meilunbio) supplemented with 10% fetal bovine serum (FBS, #F8318, Sigma‐Aldrich). NIH3T3 cells were cultured in DMEM (#MA0212, Meilunbio) with 10% FBS. All growth media were supplemented with 1% Penicillin–Streptomycin–Glutamine (#10 378 016, Gibco). Cell cultures were maintained in culture flasks in 5% CO₂ atmosphere at 37 °C. To avoid the disturbance of relevant endogenous factors in FBS, FBS were stripped with dextran‐coated charcoal (DCC) to remove most of its hormone, growth factors, and cytokines. Phenol red‐free media along with 5% DCC‐stripped serum‐starvation (CSS) were used for treatment studies.

The full length TGFB1 promoter regions, sequence 1 (0 to -500 bp region of TGFB1 promoter) and sequence 2 (-1500 to -2000 bp region of TGFB1 promoter) were cloned into the vector pGL4.18[luc2P/Neo] (Promega Corp., Fitchburg, WI, USA). An AP-1 reporter vector (pGMAP-1 Lu) with the AP-1 response element was purchased from Genomeditech, LTD (Shanghai, China). Cells were cotransfected with the indicated plasmids together with the reporter plasmid and the pGL4.74[hRluc/TK] vector using the liposome method. Cells were harvested 24 h later, and then firefly and Renilla luciferase activities were assayed with dual-luciferase assay system (Promega Corp., Fitchburg, WI, USA) according to the manufacturer's instructions. Firefly luciferase activity was normalized to Renilla luciferase activity. All experiments were, performed in triplicate, and the luminescence values for each group were statistically analysed.