Figure 5

Trio positively modulates Myh9 expression and physically interacts with Myh9 in NCCs. (A) Heat map illustrating protein level changes following Trio knockdown in NCCs. The red and blue colors indicate higher and lower relative expression of proteins in shTrio compared to shCtrl, respectively. (B) Table quantifying the number of differentially expressed proteins (DEPs) and volcano plot demonstrating downregulated (red dots on the left) and upregulated proteins (red dots on the right) between the shCtrl and shTrio groups. (C) qRT-PCR of the Myh9 mRNA level in shCtrl and shTrio NCCs (n = 5). (D) Western blot of Myh9 protein expression between shCtrl and shTrio NCCs. (E) Wound healing analysis of the shCtrl, shTrio, shMyh9, and shTrio + shMyh9 groups at 0, 12 and 24 h. Bar = 200 μm. (F) Confocal microscopy images of F-actin (green) demonstrating NCC cytoskeleton morphology in the shCtrl, shTrio, shMyh9 and shTrio +shMyh9 groups. The nuclei were counterstained with DAPI (blue). Bar = 200 μm. (G) Colocalization (yellow) of immunostained Trio (green) and Myh9 (red). Bar = 200 μm. (H) PLA detection and visualization of Trio and Myh9 in NCCs. Blue: DAPI nuclear staining. Yellow spots: PLA signals. Bar = 400 μm. (I) Domain architecture of RhoGEF Trio and Myh9. Trio-GEFD1 and the Myh9 head domain shown in red frames highlight the binding domains. (J) Pull-down analysis of potential Trio and Myh9 interactions. Bound proteins were separated by SDS-PAGE in duplicate and analyzed by western blot with anti-HIS (Histidine) and anti-GST (Glutathione S-transferase) antibodies. The red arrow at 96 kDa indicates Trio-GEFD1, the green arrow at 68 kDa indicates Myh9, and the blue arrow at 25 kDa indicates GST.