Fig. 4

The generation of OSCC OE-VGF stable cells and phenotypic influences. (A, C) SAS cell. (B, D) FaDu cell. (A, B) Lt, qPCR analysis. Rt, Western blot analysis. (C, D) Lt, growth. A two-way ANOVA test is performed to evaluate the difference in growth in the exponential growth phase between day 2 and day 4 in SAS cells and between day 3 and day 5 in FaDu cells. The original growth curves are shown in Fig. S2. Rt, cell migration assay. Drastic increases in VGF mRNA expression, supernatant VGF protein, growth, and migration are noted in stable cell subclones relative to controls. Con, vector alone control. (E – G) The effects of supernatant and co-cultivated OSCC OE-VGF. (E) The VGF localization in SAS OE-VGF cell following BFA treatment for 24 h. Upper, With the treatment of 200 ng/ml, the secretion of VGF is completely inhibited, and the cytosolic retention of VGF is noted. Lower, the gradual decrease of VGF secretion and accumulation of cytosolic VGF notified following the 0–100 ng/ml BFA treatment. (F) Upper, Western blot analysis of the supernatant was collected in two turns during the continuous SAS OE-VGF culture. Middle and Lower, growth. A two-way ANOVA test is performed to evaluate the difference in growth in the exponential growth phase between day 3 and day 5. The original growth curves are shown in Fig. S4. Adding supernatants at day 1 and day 3 to culture barely affects the growth of SAS and significantly accelerates OMF-1 growth. Con, vector alone control. (G) Upper, schematic diagram to illustrate the co-culture system. Middle, co-cultivated SAS OE-VGF increases the migration of OSCC cells compared to the control. Lower, co-cultivated OSCC OE-VGF increases the migration of OMF-1 cells compared to the control