Fig. 1: SAAs-dependent citrullination of Fbg.

a Summary model depicting the occurrence of lung metastasis at the citrullination site in cancer patients (upper). Replacement of Saa cluster genes in the mouse genome by a human counterpart (middle). Screening of genes related to the pre-metastatic phase using Fbg-deposited pulmonary vessels from noncancer and cancer patients. Generation of murine SAA gene cluster deficient and human SAA gene cluster transgene mice. Genomic structure of mouse and human SAA cluster genes (bottom). b Summary of microarray data. Venn diagram of genes with an over 4-fold increase in mRNA expressions comparing No. 1 vs. No. 4, No. 2 vs. No. 4, and No. 3 vs. No. 4 depicted in Fig. 1a. Gene names and accession numbers for No. 2 vs. No. 4 are shown in supplementary Table 1. c SAA3 and SAA1/2 proteins were colocalized with Fbg in LLC-bearing mouse lungs. Scale bar, 50 μm. d Representative immunohistochemistry (IHC) photo of SAA3 expression in endothelial cells (EC) of tumor-conditioned media (TCM)-stimulated mouse lungs. Scale bar, 50 μm. e SAA3 induction in LLC-bearing-wild-type but not -SAAs−/− mice (left) (n = 3 WT-no tumor; n = 6 WT-tumor; n = 4 SAAs−/−-no tumor; n = 6 SAAs−/−-tumor), and Fbg induction (right) (n = 6 WT-no tumor; n = 8 WT-tumor; n = 4 SAAs−/−-no tumor; n = 6 SAAs−/−-tumor). One-way ANOVA with Bonferroni correction. Mean ± SEM. f Appearance of a shifted band of Fbg in the total lysate and SAA3-IP samples prepared from primary tumor-stimulating lungs. Arrow shows a citrullinated α chain of Fbg. Two independent experiments. g Fbg bound to SAA3 in LLC-bearing lungs was assumed to be citrullinated (arrow). Immunoprecipitated SAA3 (left bottom) was also presented in the western blot. Two independent experiments. h The citrullinated Fbg band in tumor-bearings in a SAAs-dependent manner (arrow). Two anti-citrullination antibodies were used. Three independent experiments. i, j Immunohistochemical colocalization signal between citrulline and Fbg on MECA32⁺-endothelial cells in LLC-bearing mouse lungs (i) and the signal was prominent in wild-type compared to SAAs−/− mice (arrowhead). Scale bar, 20 μm. k Immunohistochemical quantification of citrulline (left) (n = 8 WT-no tumor; n = 8 WT-LLC tumor; n = 6 SAAs−/−−no tumor; n = 8 SAAs−/−−LLC tumor), Fbg (middle), and citrullinated Fbg (right) (n = 4 WT-no tumor; n = 5 WT-LLC tumor; n = 4 SAAs−/−−no tumor; n = 4 SAAs−/−−LLC tumor) in no tumor-bearing- and LLC-bearing-wild-type and SAAs-/- mouse lungs. One-way ANOVA with Bonferroni correction. Mean ± SEM.