The mutation (p. Ala515Val) in SEMA4D was a loss-of-function mutation. (A) The Sanger sequencing of SEMA4D gene in F5 family showed a homozygous mutation (p. Ala515Val) from the KS proband (F5-4). His parents (F5-1, F5-2) and brother carried the heterozygous mutation (p. Ala515Val) in SEMA4D. (B) Conservation analyses of the residue in SEMA4D protein were performed across different species, including human, chimpanzee, mouse, rat, dag, chicken and zebrafish. (C) The mutation (p. Ala515Val) was shown in the schematic diagram of SEMA4D. (D) The relative mRNA expression of human wild type and mutant SEMA4D. (E) The relative mRNA expression of mouse wild type and mutant SEMA4D. (F) The protein expression of mouse wild type and mutant SEMA4D. (G) The protein expression of mouse wild type and mutant SEMA4D in conditioned medium from COS7 cells transfected with Mouse-Sema4D-WT and Mouse-Sema4D-Ala515Val constructs. (H) Transwell assay of GN11 cells with Mouse-Sema4D-WT-CM and Mouse-Sema4D-Ala515Val-CM as lower chamber medium. (I) Scratch assay of GN11 cells treated with Mouse-Sema4D-WT-CM and Mouse-Sema4D-Ala515Val-CM. (J) P-Met staining of GN11 cells treated with HGF, Mouse-Sema4D-WT-CM, Mouse-Sema4D-Ala515Val-CM, and EV-CM.