Fig. 4.

Inhibition of the Trio RhoA-GEF domain prevents Shroom3 induced AC. (A,B) RT-PCR performed on RNA isolated from MDCK cells (A) or chicken embryo eyes from the indicated stages (B) reveals the presence of both Trio and kalirin mRNA. (C) Lens pit cells from the eye region of E10.5 transgenic mouse embryos expressing GFP (the Le-cre line) were isolated by flow cytometry. Scatter plot depicts the cell populations isolated. qPCR for lens and retina marker genes was performed on RNA from the GFP+ and GFP– cell populations. The fold expression difference is listed in the table. Both Trio and kalirin are expressed in the GFP+ and GFP– cell populations. –RT, without reverse transcriptase. (D-G) MDCK cells were transfected with the indicated plasmid(s). Where indicated, they were treated with Y27632 or G04. Immunofluorescent labeling of Shroom3-Flag (red, E) or Trio-GFP (green, G) was used to identify transfected cells. ZO1 labeling (green in E, red in G) allowed visualization and quantification of the apical area. Error bars represent s.e.m. and the asterisks identify experimental groups significantly different from the control (P<0.05). The number of cells analyzed for each experimental group is listed at the base of each bar in the histogram.