Establishing Stable Cell Lines for IL-6, STAT3, and PIAS3 Studies

Full-length human IL-6, STAT3, and PIAS3 expression vectors were constructed as previously described [26 (link),27 (link)]. Briefly, the human STAT3 (pcDNA-STAT3) and PIAS3 (pcDNA-PIAS3) expression vectors were constructed by cloning the STAT3 cDNA vector (MCG: 4909141) and the PIAS3 cDNA vector (MGC: 3528679), respectively, into the pcDNA3 expression vector (Invitrogen, Carlsbad, CA, USA). The human IL-6 expression vector (pcDNA-IL-6) was constructed by cloning the IL-6 cDNA vector (MCG: 9215) after digestion with Eco RI and Not I into the pcDNA3.1/Zeo expression vector (Invitrogen). The human HO-1 expression vector was constructed by cloning a full-length HO-1 cDNA (MGC:1723; Invitrogen) into the pcDNA3.1/Zeo expression vector (Invitrogen) with Eco R1 sites. The HO-1 or IL-6 expression vector was transiently transfected into HepG2 or Hep3B cells, respectively, by electroporation, as previously described [28 (link)]. Cells were maintained in RPMI medium with 10% FCS and 100 μg/mL of Zeocin (Invitrogen). HO-1 or IL-6 expression in resistant colonies (HepG2-HO1, Hep3B-HO1, and HepG2-IL-6) was evaluated by immunoblot, RT-qPCR, or ELISA, as described below. The mock-transfected HepG2 cells (HepG2–DNA) and Hep3B (Hep3B–DNA) were transfected with the control pcDNA3.1/Zeo expression vector and also selected by Zeocin.