Fig. 3

Induction of VGF expression in OSCC cells. (A) VGF mRNA expression in cell lines. H1299 serves as a control to denote the relative VGF expression levels in OSCC cells. (B) Western blot analysis of supernatant and cell lysate. (C) Transient exogenous VGF expression. Upper, SAS; Middle OECM1; Lower, FaDu. Lt, VGF mRNA expression following the transfection for 24 h. Rt, Western blot analysis to show the VGF protein in the supernatant and cell lysate following the transfection for 12, 24, or 48 h. Con, vector alone control; VGF, HR’-VGF plasmid. (D – G) Induction of endogenous VGF expression using SAM system. (D) Schematic diagram to illustrate 9 predicted sgRNA binding segments in VGF promoter that may allow for SAM-based activation. (E) sgRNA sequences. (F) qPCR analysis. The changes in VGF mRNA expression following SAM induction using solitary sgRNA or combined sgRNAs transfection. The combination of sgRNA 109/133/154/179 yields induction effects simulating the combination of 8 sgRNAs. (G) Lt, qPCR analysis. Rt. Western blot analysis. It shows that the induction of VGF mRNA expression is associated with the increased VGF protein in the supernatant 24 h after induction. Con, vector alone control