Lentiviral vectors small hairpin rna shrna

The lentiviral vectors (LV)-small hairpin RNA (shRNA) based on a third generation lentiviral system targeting METTL3 (LV-shMETTL3) and negative control (LV-shNC) were purchased from Shanghai GeneChem Co., Ltd. Briefly, 2.0 µg shRNA vector and 2.0 µg packaging mix plasmids were transfected into 293T cells (cat. no. C6008; Beyotime Institute of Biotechnology) using Lipofectamine 3000^® reagent (Thermo Fisher Scientific, Inc.) for 24 h at 37°C, and the medium was then harvested and the virus was extracted by 85,000 × g ultracentrifugation at 4°C for 2 h in a centrifuge (Beckman Coulter, Inc.). For lentiviral transduction, the cells were seeded in six-well plates at a density of 50,000 cells/well. The lentiviral vector was added at a multiplicity of infection of 20, with 8 µg polybrene (Sigma-Aldrich; Merck KGaA) per well. After 72 h, the cells infected with the lentiviral vectors were selected using puromycin (Beyotime Institute of Biotechnology). The concentration of puromycin used for selection was 2 µg/ml, and the concentration used for maintenance was 1 µg/ml. The knockdown efficiency was evaluated using RT-qPCR.

The lentiviral vectors (Lv)-small hairpin RNA (shRNA) based on a 3rd generation lentiviral system targeting PLAU (Lv-shSPP1), Lv-shYY1, and negative control (Lv-shCon) were purchased from Shanghai GeneChem Co., Ltd. Briefly, 1.5 µg shRNA vector and 1.5 µg package mix plasmids were transfected into 293T cells (cat. no. C6008; Beyotime Institute of Biotechnology) with Lipofectamine 3000^® reagent (cat. no. L3000001; Thermo Fisher Scientific, Inc.) for 24 h at 37°C, and the medium was then harvested and the virus was extracted by 80,000 × g-ultracentrifugation at 4°C for 2 h in a centrifuge (Beckman Coulter, Inc.). For lentiviral transduction, the cells were seeded in six-well plates at a density of 50,000 cells/well. The lentiviral vector was added at a multiplicity of infection of 15, with 5 µl polybrene (Sigma-Aldrich; Merck KGaA) per well. After 72 h, cells infected with the lentiviral vectors were selected using 1 µg/ml puromycin (Beyotime Institute of Biotechnology). Knockdown efficiency was evaluated using RT-qPCR.