Generation of stable cell lines and transient transfection

FZ Fuxing Zhou
XY Xiaoshan Yang
HZ Hang Zhao
YL Yu Liu
YF Yang Feng
RA Rui An
XL Xiaohui Lv
JL Jia Li
BC Biliang Chen
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A2780 (50,000 cells/well) and SKOV3 (80,000 cells/ well) cells were cultured in 6-well plates for 18 h before being treated with lentiviral vectors for 8 h. The cells were incubated further in normal culture medium for 48 h, and then, puromycin was added for 3-4 weeks to establish stable cell lines. The cDNA encoding SNAP-29, SNAP-29 (Mut) and LC3 were cloned in pcDNA3.1, pEGFP-N1 or pmCherry-C1 vectors. The cells were transfected with Lip2000 (Invitrogen, 11668019). The siRNA sequences were: SNAP-29, 5'-AGACAGAAAUUGAGGAGCA-3'; Stx17, 5'- CCGAAAGGAUGACCUAGUA-3'; VAMP8, 5'-GCAACAAGACAGAGGAUCU- 3'. The siRNA was delivered into cells with Lipo2000 according to the instructions of the manufacturer.

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