Extended Data Figure 7: DR6 is expressed in ECs, and DR6 expressed in immune cells is not involved in TC metastasis.

a, Western blot analysis of lysates from HUVECs transfected with scrambled siRNA (siCTRL) or different sets of siRNAs directed against mRNA encoding DR6 (siDR6). The antibody detects the 90 kDa glycosylated form of DR6. α-tubulin served as loading control. b, Expression levels of DR6 in different human or mouse ECs as determined by quantitative PCR. Shown are relative expression levels normalized to GAPDH levels. c, Confocal images of human tissues of the indicated origin stained for CD31 (red), DR6 (green) and cell nuclei (DAPI, blue) (left panel). Control-IgG antibody and donkey anti-rabbit secondary antibody coupled to AF488 served as negative controls (right panels); scale bar, 5 μm. d, Analysis of HUVEC single-cell gene expression for DR6 as determined by the 2^LoDct-ct method and normalized to the cell with the highest expression level (100%). Each bar represents the gene expression level of one individual cell (data of a total of 80 cells analysed are shown). Single-cell analysis revealed that less than 10% of ECs express DR6. e–j, Irradiated DR6^+/+ or DR6^−/− animals were reconstituted with bone marrow cells from DR6^+/+ or DR6^−/− donor animals, respectively (DR6^+/+ → DR6^+/+, DR6^−/− → DR6^+/+ or DR6^+/+ → DR6^−/−) and quantitative PCR analysis of DR6 expression in PBMCs was performed (e) or bone marrow chimaeras as indicated were injected i.v. with B16 TCs and lungs were analysed after 6 h for pulmonary EthD-III-positive ECs (g) and extravascular TCs (h) or after 12 d for lung metastases (j). f, i, Representative confocal images of lung sections stained for the indicated markers (f) and images of whole lungs (i); scale bar, 50 μm. Shown are representative data of two independent experiments with mean values ± s.d. (e, j) or ± s.e.m. (g, h) from n = 3 (e) or n = 4 (g, h) animals per condition or n = 6 (DR6^+/+ → DR6^+/+), n = 4 (DR6^−/− → DR6^+/+) and n = 5 (DR6^+/+ → DR6^−/−) animals (j). **P < 0.01; n.s., not significant. One-way ANOVA and Bonferroni’s post hoc test. For gel source data see Supplementary Fig. 1.