*2.5. Plasmid and Transfection of MSC Culture*

We used the pcNS3-NS5B plasmid construct encoding five nonstructural HCV proteins (NS3, NS4A, NS4B, NS5A, and NS5B) of genotype 1b that was constructed using a commercially available pcDNA-3.1(+) vector (Invitrogen, USA) [30]. The plasmid was purified from *E. coli* strain *JM109* using a commercial QIAGEN Plasmid Purification Maxi Kit (QIAGEN, Hinden, Germany) according to the manufacturer's instructions.

To obtain genetically transformed cells expressing HCV proteins, we used a primary MSC culture at third-fourth passages. MSCs were seeded on a six-well plate at a density of 5x10<sup>4</sup> cells/mL. Twenty-four hours after reaching the subconfluent monolayer (70–90% cells/well), complexes of a plasmid with Xfect Transfection Reagent (Clontech Laboratories, Takara, USA) were applied to the cells. The transformed cells were selected in a medium containing 0.5 mg/mL G-418 (Invitrogen, Waltham, MA, USA). Cell viability was analyzed using a standard MTT test [31] and the trypan blue dye exclusion assay [32]. We conducted several rounds of selection, changing the medium with G-418 every 72 h. Cytokine secretion was measured by quantifying their levels in the conditioned medium.
