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. 2020 Sep 23;133(18):jcs243683. doi: 10.1242/jcs.243683

Fig. 6.

Fig. 6.

Rabgap1 enhances invasive migration in vitro and high Rabgap1 expression correlates with poor prognosis of breast cancer patients. (A,B) Quantification of total (A) and active (B) α5β1 integrin recycling in control (shCtr) or Rabgap1-depleted MDA-MB-231 cells (shRabgap1) by capture ELISA. Data are mean±s.e.m. n=4. **P<0.01; n.s., not significant (unpaired t-test). (C) Confocal microscopy analysis of control (shCtr) and Rabgap1-depleted (shRabgap1) MDA-MB-231 cells migrating into a fibronectin-supplemented Matrigel 3D matrix in an inverted invasion assay. Cells were stained for F-actin using phalloidin–TRITC, and nuclei were labeled with DAPI. Serial optical sections were captured at 15 µm intervals and are presented as a sequence in which the individual optical sections are placed alongside one another, with increasing depth from left to right. Invasion is expressed as the proportion of cells that migrate further than 45 μm (red line). Scale bar: 20 µm. (D) Quantification of the invasiveness of control (shCtr) and Rabgap1-depleted (shRabgap1) MDA-MB-231 cells using the area calculator plugin in ImageJ. The fluorescence intensity of cells invading more than 45 μm was measured and displayed as a percentage of the fluorescence intensity of all cells within the plug. Data are mean±s.d.; n=15 (shCtr) and n=12 (shRabgap1). *P<0.05 (unpaired t-test). (E) Representative confocal images of invading control and Rabgap1-depleted MDA-MB-231 cells stained with phalloidin–TRITC and DAPI to label F-actin and nuclei, respectively. Scale bar: 10 µm. (F) Quantification of pseudopod length of invading control and Rabgap1-depleted MDA-MB-231 cells from confocal images. Data are mean±s.d. n=38. ***P<0.001 (unpaired t-test). (G) Kaplan–Meier analysis indicates a decreased disease-free survival of breast cancer patients with high Rabgap1 expression levels. The analyses show that individuals with high Rabgap1 expression display a significant increase in the hazard ratio (HR) and a pronounced increase in the disease risk compared with individuals with low Rabgap1 expression levels.