Figure 7. EGF induces ntcp-sfGFP containing ruffling that is blocked by inhibition of PKCζ.

Ntcp-sfGFP expressing Huh7 cells were cultured on glass-bottomed chambers and imaged under live cell fluorescence microscopy. A. At time 0, EGF (100 ng/ml) was added to the chamber and cells were imaged in the ntcp-sfGFP channel for an additional 15 min at 1 frame per min. At time points prior to EGF addition ruffles are not seen. Yellow lines indicate ntcp-sfGFP containing ruffles that form and move centripetally across cells before being absorbed (supplementary movie 1). B. In the presence of 12.5 µM PKCζ pseudosubstrate inhibitor (EGF+PS), ruffle formation is inhibited. Time in minutes is at the lower left of each panel (supplementary movie 2). C. Higher magnification imaging reveals that as ruffles are absorbed into the cell, small extensions containing ntcp form throughout the cell. Duplicate images with lines drawn highlight a ruffle reabsorbing into the cell (yellow line) and new extensions (smaller yellow lines; supplementary movie 3). D. Representative images demonstrate Ntcp-sfGFP-containing ruffle induction for ligands of three receptor tyrosine kinases, Ctl (buffer alone), EGF (100 ng/mL), insulin (20 ug/mL), PDGF (500 ng / mL). EGF induced substantial ntcp containing ruffles while insulin induced some ruffles, but PDGF and buffer alone did not alter ntcp distribution.