Extended Data Figure 6: Effects of zVAD treatment and EC-specific loss of caspase–8 on metastasis formation.

a, Western blot analysis of uncleaved caspase-8 (Casp8) and VE-cadherin (VE-Cad) in primary MLECs of tamoxifen-treated Tie2-CreER^T2;Casp8^loxP/loxP animals (Casp8^ECKO). b, Quantification of Evans blue permeability in the lungs of Casp8^ECKO animals. c, Quantification of transmigrated B16 or LLC1 TCs over a layer of DMSO- or 4-OH-tamoxifen-treated primary MLEC from uninduced Tie2-CreER^T2;Casp8^loxP/loxP animals or Cre-negative control littermates. d, e, Quantification of lung metastases 12 d after i.v. injection (d) or 27 d after excision of a primary tumour induced by s.c. injection (e) of B16 or LLC1 TCs into Casp8^ECKO animals. Cre-negative littermates served as control. No significant differences in primary tumour growth were observed (data not shown). f–h, Effect of z-VAD-fmk (zVAD, 100 μM) on B16 TC proliferation (f), viability (g) and migration (h). i, Representative confocal images of lung sections taken 6 h and images of whole lungs taken 12 d after i.v. injection of B16 TCs into WT animals treated with DMSO (control) or zVAD shortly before and at 3 h after TC injection (plus at 6 h for the 12 d experiment); scale bar, 50 μm. Shown are representative data of two (b, c, e) or three (d, f–h) independent experiments with mean values ± s.d. (b–e, h) or ± s.e.m. (f, g) from n = 3 (b) or n = 5 (d) animals per group or from n = 11 (B16, control), n = 7 (B16, Casp8^ECKO), n = 8 (LLC1, control) and n = = 6 (LLC1, Casp8^ECKO) animals (e) or from biological sextuplicates (n = 6) (c, f, g) or triplicates (n = 3) (h). *P < 0.05; ***P < 0.001; n.s., not significant. Unpaired, two-tailed Student’s t-test (b, d–h) or one-way ANOVA and Bonferroni’s post hoc test (c). For gel source data see Supplementary Fig. 1.