Supplemental Figure S1
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Fig. S1. Expression of HAI2 affects HGF-induced scattering but not proliferation. apoptosis and size of MCDK type I cells. (A) Quantification of HGF-induced scattering of MDCK II cells (see Fig. 1A-F). Treatment with 100 ng/ml of active HGF or pro-HGF dramatically increased the number of cells in single-cell colonies (blue) and fragmented small microcolonies that contained two to five cells (purple), whereas the proportion of cells in more intact colonies containing 6-20 (green) and >21 (brown) cells was correspondingly reduced. The addition of 20 nM or 60 nM of human recombinant HAI2 strongly inhibited pro-HGF-induced fragmentation of MDCK II colonies. (B-F) Effect of HAI2 expression on proliferation, survival and size of MDCK I cells. (B) Proliferation of MDCK I cells expressing full-length human recombinant HAI2 protein (green) and control cells (blue) in culture. The numbers of cells is normalized to the values at day 1 (0 hours). The proliferation rate of HAI2-expressing cells was comparable to that of control cells. (C) Apoptotic rates of MDCK I cells during the acquisition of transepithelial electrical resistance (TEER). Caspase 3 activity in protein lysates from MDCK I cells expressing full-length human recombinant HAI2 protein (green) and control cells (blue) at 4 days (onset of tight junction formation, left) and 7 days (establishment of functional epithelial barrier, right) after seeding on Transwell filters. No effect of HAI2 expression on the apoptotic rate at either stage was observed. (D-F) Statistical distribution (D,E) and geometric mean (F) of cell size of MDCK I cells expressing full-length human recombinant HAI2 protein and control cells 4 days (D) and 7 days (E) after seeding in duplicate on Transwell filters, as determined by flow cytometry. HAI2-expressing cells (F, green) were on average ∼6% larger at day 4 (F, left) and 10% larger at day 7 (F, right) than the control cells (F, blue).
