Puromycin

The pLV-hsa-miR-1 plasmid and the negative control pLV-miRNA-vector were purchased from Shanghai GenePharma Co., Ltd. The viruses were packaged in 293T (Type Culture Collection of the Chinese Academy of Sciences) cells according to standard protocols and the virus particles were harvested 72 h later. The packaged lentiviruses were termed LV-miR-1, while the empty lentiviral vector LV-ctrl was used as a control. Cells were infected with viral particles (MOI = 10 for each) for 24 h and 5 µg/ml Polybrene, which was followed by selection with puromycin (2 µg/ml) for ≤7 days. Polybrene and puromycin were purchased from Shanghai GenePharma Co., Ltd.

BRD4-targeted shRNA was expressed in a pGLV3/H1/green fluorescent protein (GFP) + Puro lentiviral vector via BamHI and EcoRI; vectors were constructed by Shanghai GenePharma Co., Ltd. (Shanghai, China). The shRNA sequence targeting BRD4 was as follows: 5′-GCTCAAGACACTATGGAAACA-3′, the shRNA sequence used as the negative control (sh-NC) was 5′-TTCTCCGAACGTGTCACGT-3′ (LV3-NC-GFP; Shanghai GenePharma Co., Ltd.). 293 cells (American Type Culture Collection) were cultured in 10 cm plates at a density of 5×10⁵ cells per well with DMEM (Gibco; Thermo Fisher Scientific, Inc.) containing 10% FBS in an incubator at 37°C and 5% CO₂ for 48 h. Then lentiviral shRNA-containing plasmids were transfected into 293 cells to generate lentiviruses; the lentiviral supernatant containing the BRD4 shRNA was harvested and purified. DU145 and LNCAP cells were then infected by application of viral supernatant (multiplicity of infection 15 for DU145, multiplicity of infection 20 for LNCAP) in RPMI-1640 culture medium with 10% FBS. The cationic polymer Polybrene (Shanghai GenePharma Co., Ltd.) was added to facilitate transduction. After 24 h following transduction the medium was exchanged and cells were cultured for an additional 48 h. Stable cell lines were selected via the application of 1 µg/ml puromycin (Shanghai GenePharma Co., Ltd.).

PC-3 human PCa cells from the Cell Bank of Xi'an Jiaotong University were cultured in medium containing 10% fetal bovine serum (Hyclone Laboratories; GE Healthcare Life Sciences), 1% streptomycin mixture and ~90% RPMI-1640 complete medium (Gibco; Thermo Fisher Scientific Inc.). The cells were cultured at a constant temperature of 37°C with 95% O₂, and 5% CO₂ in an incubator (relative humidity, ~95%). Adherent cells were transfected with GAB2 siRNA as follows: The commercial LV3-GAB2 lentivirus vector (containing green fluorescent protein and the puromycin sequence) and GAB2 siRNA sequences were constructed by Shanghai GenePharma Co., Ltd. The sequence of GAB2 siRNA was 5′-GGGACCTCCTGGTAGACAATA-3′, and the sequence of scrambled fluorescein-labeled negative control siRNA was: 5′-TTCTCCGAACGTGTCACGT-3′. When cells reached 40–50% confluence in the presence of 8 µg/ml polypropylene, lentiviral vectors were used to infect PC-3 cells at 50 multiplicity of infection for 72 h. The stable GAB2 low expression level subclones were maintained via 2–3 µg/ml puromycin-resistant culturing (puromycin, Sigma-Aldrich; Merck KGaA). PC-3 human PCa cells transfected with negative control (NC) siRNA were used as the NC group, and cells transfected with GAB2 siRNA were used as the knockdown (KD) group. Three samples in each group were subjected to gene chip analysis.