Fig. 4. Expression of TG2 mutants in SCC13-TG2-shRNA2 cells.

A TG2 mutants and impact on TGase and GTP-binding/G-protein-related activity. B SCC13-TG2-shRNA2 cells (TG2 knockdown) were electroporated with plasmids encoding wild-type or mutant TG2, or empty vector (EV) and grown as monolayers in spheroid growth medium. After three days, the cells were harvested for immunoblot with anti-TG2. C SCC13-TG2-shRNA2 cells were electroporated with plasmids encoding the indicated plasmids, plated on attachment plates in spheroid medium and after 3 d the cells were fixed, permeabilized and stained with anti-TG2. As a control, SCC13-Control-shRNA cells were electroporated with empty vector and stained in parallel. TG2 detected in these cells is endogenous. Similar findings were observed in each of three repeated experiments. D SCC13-Control-shRNA cells were electroporated with 3 μg of empty vector (EV) and SCC13-TG2-shRNA2 cells were electroporated with plasmid encoding TG2-wt, TG2 mutants (C277S, R580A, Y526F or W241A) or empty vector. After electroporation, the cells were seeded at 40,000 cells in each of six low-attachment wells in 2.5 ml of spheroid media. Spheroids were counted on days 1, 3 and 5.