Figure 6. Vinculin downstream of ERα is important for metastasis.

MCF-7-luc2 cells and CRISPR/Cas9-mediated VCL-deleted MCF-7-luc2 cells were injected into nude mice via the tail vein to generate xenografts (n=5). (a) Bioluminescence imaging of the control or the Cas9-vinculin group. Tumour formation in the lung was determined using H&E staining. (b) Luciferase counts in the lungs of mice on week 4. (c) Representative confocal images of mouse lungs 0.5 and 24 h after tail vein co-injection of control MCF-7 cells (red) and Cas9-vinculin MCF-7 cells (green). Scale bar, 100 μm. (d) Quantification of cells retained in the lung after tail vein injection (n=4 mice). (e) A transwell assay was performed in ERα-overexpressing MDA-MB-231 cells infected with lentivirus containing vinculin short hairpin RNA (shRNA; sh-VCL) or scrambled RNA (scramble; n=3). Control MDA-MB-231-luc2 cells, GM6001-treated control cells or GM6001-treated and CRISPR/Cas9-mediated VCL-deleted MDA-MB-231 cells were injected into nude mice to generate xenografts (n=5). (f) Representative bioluminescence images of different groups on week 4 are shown. The cell morphology of the primary tumour was determined by H&E staining. Scale bar, 50 μm. The particular section of H&E image was enlarged to highlight the cell morphology. (g) Luciferase counts of metastasis sites on week 4. (h) Roundness index of corresponding MDA-MB-231 cells from the primary tumour in f (n=200 cells). (b,d,e,g,h) The data are shown as the mean±s.e.m. ns P>0.05, **P<0.01, ***P<0.001. (b,d,e) Unpaired t-test; (g,h) ANOVA with Tukey's post hoc test.