Fig. 8: The N-terminal region of Rab26 is crucial for regulating migration/invasion of breast cancer cells.

A Rab26 has an extra N-terminal extension in amino-acid sequence compared with other Rab proteins. B Wound-healing assays showed over-expression of Rab26b (which does not contain the N-terminal extension) did not inhibit migration of MDA-MB-231 cells. C Quantitative analysis of the wound area using imageJ software from three independent wound-healing experiments, *p < 0.05. D Matrigel transwell invasion assay showed over-expression of Rab26b does not inhibit invasion of MDA-MB-231 cells. E Quantitative analysis of C from three independent experiments, *p < 0.05. F Double-layer soft-agar experiments showed that over-expression of Rab26b does not inhibit colony formation of MDA-MB-231 cells in soft agar. G Rab26b does not interact with ATG16L1 in GST pull-down assay. H GFP-Rab26b is distributed in cytosol, not associating with mCherry-ATG16L1 in MDA-MB-231 cells, bar = 20 μm. I Rab26b does not induce the degradation of phosphorylated Src in western-blot experiments. J Quantitative analysis of the results of I from three independent experiments, *p < 0.05. K A model for Rab26 inhibiting cell migration/invasion through mediating autophagic degradation of phosphorylated Src. In this model, Rab26 associates with endosomes and recruits ATG16L1 to generate autophagophore, and subsequent LC3 to autophagosomal isolate membrane, LC3 then recruits the active Src (p-Src). After autophagosome maturation, it fuses with lysosome to degrade p-Src. The degradation of the active Src may inhibit formation or turnover of focal adhesion, consequently inhibiting cell migration and invasion.