2.7. Transfection of shRNA and plasmid DNA

The short hairpin RNA (shRNA) of c-Jun and its negative control were described as follow, the shRNA sequences of c-Jun were as follows: c-Jun shRNA (target sequence: 5′-GCAGCAGCAGCCGCCGCACCA-3′) and control shRNA (target sequence: 5′-CAACAAGATGAAGAGCACCAA-3′). Cells were cultured in a 6-well plate in a density of 3×10⁵ cells/well under media conditions. Then the cells were transfected with c-Jun shRNA or plasmid DNA in lipofectamine 2000 according to the manufacturer׳s instructions, the final concentration of c-Jun shRNA and the c-Jun plasmid was 4 μg/well. Cells were also transfected with lipofectamine 2000 containing control shRNA or scramble vector as a negative control. c-Jun shRNA and control shRNA lentiviral particles used to transfect S1 and S1-MI-80 cells were collected from viral packaging 293 T cells. The positive cells, transfected with c-Jun shRNA lentivirus, were sorted after puromycin (10 μg/mL) treatment.