Extended Data Figure 4: Reduced TC-induced endothelial necroptosis and metastasis in MLKL^−/− mice.

a, A TALEN pair targeting the indicated sequences in the second exon of the mouse Mlkl gene was cloned and transcribed into mRNA. b, Analysis of mice born after mRNA injection into fertilized oocyte injection by PCR for exon 2 (top) and subsequent sequence specific endonuclease assay (T7EI, bottom) for the detection of mutant alleles. DNA from C57BL/6 (WT) or TALEN-transfected cells (mut) served as control. c, The mutant allele of mouse 64 was further analysed using Sanger sequencing revealing a 8 bp deletion (Δ8), predicted to generate a premature stop codon. d, Spleen and lung extracts of three B6D2F1 MLKL^+/+ WT and three homozygous MLKL mutant mice (MLKL^−/−) were probed for Mlkl protein expression. Polyclonal MLKL antibody detected Mlkl protein only in WT extracts but also an additional non-specific band of similar size. e–i, B6D2F1 MLKL^+/+ WT or MLKL^−/− animals were injected i.v. with B16 TCs and lungs were analysed after 6 h for pulmonary EthD-III-positive ECs (f) and extravascular TCs (g) or after 12 d for lung metastases (i). Representative confocal images of lung sections and images of whole lungs are shown in (e, h); scale bar, 50 μm. Shown are representative data of two independent experiments with mean values ± s.e.m. (f, g) or ± s.d. (i) from n = 3 animals per condition (f, g) or n = 8 (MLKL^+/+) and n = 11 (MLKL^−/−) animals (i). **P < 0.01; ***P < 0.001. Unpaired, two-tailed Student’s t-test. For gel source data see Supplementary Figs 1 and 2.