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. 2022 Jun 11;10(1):65–68. doi: 10.1016/j.gendis.2022.05.030

Figure 1.

Fig. 1

SEMA4D acts as a novel oligogenic pathogenic gene of IHH through the PlexinB1/MET/RND1/RHOA/RAF1/MAPK signaling axis. (A) The KS proband (F5-4) carried a homozygous mutation (p. Ala515Val) in SEMA4D inherited from his parents (F5-1, F5-2) who carried the heterozygous mutation (p. Ala515Val) in SEMA4D without clinical manifestations. His brother, F5-3, harbored the heterozygous mutation (p. Ala515Val) in SEMA4D with anosmia, but no hypogonadism. KS, Kallmann syndrome. (B) The mutation (p. Ala515Val) was mapped onto the ribbon diagram of the SEMA4D/PlexinB1 complex (PDB, 3OL2). The SEMA4D protein was colored as green, and the PlexinB1 was colored as magenta. The mutant residue and relevant residues were shown as stick in different color. (C) The protein expression of human wild type and mutant SEMA4D. (D) The protein expression of human wild type and mutant SEMA4D in conditioned medium from 293T cells transfected with Human-SEMA4D-WT and Human-SEMA4D-Ala515Val constructs. (E) Migrated cells in Transwell assay were presented through bar graph. Data are expressed as mean ± standard deviation (n = 3 per group). ∗P < 0.05. (F) Actin and DAPA staining showed the morphology of GN11 cells treated with HGF, Mouse-Sema4D-WT-CM, Mouse-Sema4D-Ala515Val-CM, and EV-CM after serum starve pretreatment. Scale bar = 20 μm. EV, empty vector; WT, wild type; CM, conditioned medium. (G) Pull-Down assay of SEMA4D and PlexinB1 with Input and IgG as control. (H–J) Migrated cells in Transwell assay of GN11 cells transfected with si PlexinB1, si Met and si Rnd1 were presented through bar graph respectively. Data are expressed as mean ± standard deviation (n = 3 per group). ∗P < 0.05, ∗∗P < 0.01 and ∗∗∗P < 0.001. (K) Representative Western blot results for PlexinB1, P-Met, Met, GTP-Rnd1, GTP-RhoA, P-Raf1, Raf1, P-Erk1/2 and Erk1/2 in GN11 cells treated with HGF, Mouse-Sema4D-WT-CM, Mouse-Sema4D-Ala515Val-CM, and EV-CM in the first column from left. Representative Western blot results for PlexinB1, P-Met, Met, GTP-Rnd1, GTP-RhoA, P-Raf1, Raf1, P-Erk1/2 and Erk1/2 in GN11 cells transfected with si PlexinB1, si Met, si Rnd1, si RhoA and si Raf1 in Mouse-Sema4D-WT-CM and Mouse-Sema4D-Ala515Val-CM treatment groups from the second column on the left to the right respectively. (L, M) Migrated cells in Transwell assay of GN11 cells transfected with si RhoA and si Raf1 were presented through bar graph. Data are expressed as mean ± standard deviation (n = 3 per group). ∗P < 0.05, ∗∗P < 0.01 and ∗∗∗P < 0.001. (N) The verification of genotype of experimental mice models. Only one band of 633 bp indicated Sema4D−/− genotype. Only one band of 323 bp indicated Sema4D+/+ genotype. Two bands of 633 bp and 323 bp indicated Sema4D−/+ genotype. (O) Representative Western blot results for SEMA4D in the experimental mice models. (P) The immunofluorescence staining of brain tissue in sagittal plane in new born Sema4D+/+ and Sema4D−/− mice models. DAPI, blue; Actin, Green; GnRH, Red. Scale bars = 100 μm. (Q) The immunofluorescence staining of hypothalamus tissue in new born experimental mice models. DAPI, blue; GnRH, Red. Scale bars = 20 μm. (R) The immunohistochemically staining result of GnRH in olfactory bulb and hypothalamus in E14.5 and new born experimental mice models. (S) The concentration of serum testosterone, wet weight of testis, wet weight of seminal vesicle, the progressive motility of sperm, the non-progressive motility of sperm, the immotility of sperm and the concentration of sperm in adult male mice models respectively (n = 3 per group). ∗P < 0.05, ∗∗P < 0.01 and ∗∗∗P < 0.001.