Human HCC cell lines including Huh7, SMMC-7721, Hep3B and HepG2, and the human immortalized normal hepatocyte cell line LO2 were purchased from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China) and maintained in Dulbecco's modified Eagle medium (DMEM, Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS, Gibco) with 100 units/mL penicillin and 100 μg/mL streptomycin (Sigma, St-Louis, MO, USA) and cultured in a humidified 5% CO2 incubator at 37°C.
Retroviral vectors pMMP-ZBTB20 and pMMP-FoxO1 were generated by inserting the respective cDNA into pMMP. The retroviruses were packaged and tranfected into HCC cells as previously described [36 (link)]. The targeted sequences for ZBTB20 siRNA (5′-CUA UGC GAU UAC GAC UAA GU-3′), FoxO1 siRNA (5′-GCA AAG AUG GCC UCU ACU U-3′) and a nonspecific duplex oligonucleotide as a negative control were synthesized by Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China). Cells were transfected with the vectors mentioned above using Lipofectamine 2000 according to the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA).
Retroviral vectors pMMP-ZBTB20 and pMMP-FoxO1 were generated by inserting the respective cDNA into pMMP. The retroviruses were packaged and tranfected into HCC cells as previously described [36 (link)]. The targeted sequences for ZBTB20 siRNA (5′-CUA UGC GAU UAC GAC UAA GU-3′), FoxO1 siRNA (5′-GCA AAG AUG GCC UCU ACU U-3′) and a nonspecific duplex oligonucleotide as a negative control were synthesized by Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China). Cells were transfected with the vectors mentioned above using Lipofectamine 2000 according to the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA).