Lentiviral shrna vectors

Huh-1, Huh7, PLC/PRF/5, and Hep3B are human HCC cell lines. The pLPCX-GLS2 retroviral vector which expresses GLS2 was constructed by PCR amplification as described [11 (link)]. The pLHCX-DN-AKT retroviral vector expressing a dominant negative AKT (DN-AKT; K179M) was constructed by subcloning the DN-AKT DNA fragment from pLNCX-AKT1 K179M (Addgene) into the pLHCX vectors [22 (link)]. Two lentiviral shRNA vectors against GLS2 (ID: V3LHS-307701; V2LHS-71048) and control shRNA vectors were obtained from Open Biosystems (Huntsville, AL). To establish cell lines with stable GLS2 knockdown, cells infected with shRNA vectors were selected by puromycin. For 5-Aza-2’-deoxycytidine (5-AZA-dC) treatments, cells were treated with 5 μM 5-AZA-dC (Sigma) for 7 days and 5-AZA-dC was replaced every day. Control cells were treated with DMSO.

The pLKO.1-lentiviral shRNA vectors that target Kdm5a and nonsilencing pLKO.1 control vector (Scrsh) were synthesized by Open Biosystems (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The lentiviruses were packaged via the co-transduction HEK293T cells with the lentiviral vector plasmid containing shRNA and the packaging plasmids psPAX2 and pMD2.G using Lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, CA, USA). Forty-eight hours after transfection, the viral supernatant was collected and used for the infection of MSCs.