Fig 1: Possible mechanisms for CXCR2-MSCEGFP alleviation of IBD.(A) RNA-seq data analysis of MSCs from 6 donors (3 were stimulated by IFN-γ). The heatmap displays genes encoding semaphorin proteins. This heatmap was drawn with an RPKM value of log2. (B) Western blot displaying the expression of sema7A protein in MSC, MSCEGFP, and CXCR2-MSCEGFP; a representative blot is shown. (C) Flow cytometric analysis of sema7A protein on the cell surface of MSCs from the 3 mRNA groups. All of the experiments were repeated at least 3 times. (D) The culture medium from MSCs was collected; sema7A protein was detected by ELISA (means ± SDs). (E) Sema7A mRNA expression level in MSCs assessed by quantitative real-time PCR analysis (means ± SDs). GAPDH was used as an internal mRNA control. (F) Macrophages were co-cultured with MSCs, siRNA-sema7A MSCs, or 40 nM hsema7A. The culture media were used to detect dissociated IL-10 by ELISA. (G) Cells from (F) were collected to detect the intracellular level of IL-10 by flow cytometric analysis. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.
Fig 2: Regulation of SEMA7A during inflammation.Endothelial HMEC-1 and alveolar epithelial A549 cells were exposed to pro-inflammatory cytokines A) SEMA7A immunofluorescence in endothelial HMEC-1 following stimulation with TNF-α (100ng/ml) and IL-6 (20ng/ml) for 4 hours with SEMA7A (green), Actin (red), cell DNA marker DAPI (blue) and merge (yellow). B) Relative change in SEMA7A-mRNA and protein expression after exposure of HMEC-1 cells to TNF-α and IL-6 for the indicated time periods. C) Relative change in SEMA7A-mRNA and protein expression after exposure of HMEC-1 cells to increasing concentrations of TNF-α and IL-6. D) SEMA7A immunofluorescence in epithelial A549 cells following stimulation with TNF-α and IL-6 for 4 hours with SEMA7A (green), Actin (red), cell DNA marker DAPI (blue) and merge (yellow). E) Relative change in SEMA7A-mRNA and protein expression after exposure of epithelial A549 cells to TNF-α and IL-6 for the indicated time periods. F) Relative change in SEMA7A-mRNA and protein expression after exposure of epithelial A549 cells to increasing concentrations of TNF-α and IL-6 (all the data are expressed as the mean±SEM, *P < 0.05; **P < 0.01; ***P < 0.001 as indicated, n≥4/ group, one representative picture/ blot of three is demonstrated).
Fig 3: SEMA7A induces the expression of pro-inflammatory cytokines.Endothelial HMEC-1 and alveolar epithelial A549 cells were exposed to human recombinant SEMA7A. A) TNF-α and IL-6 immunofluorescence in endothelial HMEC-1 following stimulation with SEMA7A for 4 hours, TNF-α or IL-6 (green), Actin (red), cell DNA marker DAPI (blue) and merge (yellow). B) Relative change in TNF-α and IL-6 mRNA after exposure of HMEC-1 cells to SEMA7A at different time points. C) Relative change in TNF-α and IL-6 protein expression after exposure of HMEC-1 cells to SEMA7A for up to 24 hours. D) TNF-α and IL-6 immunofluorescence in epithelial A549 cells following stimulation with SEMA7A for 4 hours, TNF-α or IL-6 (green), Actin (red), cell DNA marker DAPI (blue) and merge (yellow). E) Relative change in TNF-α and IL-6 mRNA after exposure of epithelial A549 cells to SEMA7A at different time points. F) Relative change in TNF-α and IL-6 protein expression after exposure of A549 cells to SEMA7A for up to 24 hours (all the data are expressed as the mean±SEM, *P < 0.05; **P < 0.01; as indicated, n≥4/ group, one representative picture/ blot of three is demonstrated).
Fig 4: Role of NF-κB in the regulation of SEMA7A.A) Graphic representation of the putative SEMA7A promoter. Four potential NF-κB binding elements were identified in the upstream sequence relative to the transcription start site (TSS). B) ChIP assay was employed to examine NF-κB binding to the human SEMA7A promoter in A549 monolayers after exposure to TNF-α. C) A549 cells were transfected with the SEMA7A reporter plasmid and site-directed mutation of each NF-κB binding site was performed. Empty reporter vector (pGL4.17) was used as a negative control and SEMA7A full length (SEMA7A-NF-κBFL) represents the SEMA7A promoter construct without site-directed mutation. All data are expressed as mean±SEM compared to SEMA7A-NF-κBFL, ***P < 0.001 as indicated, n = 3/ group, one representative blot of three is demonstrated).
Fig 5: Sema7A(−) CXCR2-MSCEGFP exert unsatisfactory therapeutic effect(A) Experimental mice were i.v. injected with 1 × 106 CXCR2-MSCEGFP or sema7A (−)CXCR2-MSCEGFP on day 1 of IBD. Representative photographs of colon samples from the mice of each group are displayed. (B) Colon lengths from (A) were measured (n = 3 mice/group). (C) Mice of each group were weighed from day 1 presensitization to 3 days post-sensitization (n = 3 mice/group). (D) Disease activity index of colon tissues obtained from each group during IBD (72 h post-injection). Data are presented as means ± SDs for individual mice. (E) Representative H&E images of colon sections of CXCR2-MSCEGFP-treated, and sema7A(−)CXCR2-MSCEGFP-treated mice at 24 h post-injection. Histopathological scores of colon tissue from CXCR2-MSCEGFP-treated and sema7A(−) CXCR2-MSCEGFP-treated mice are presented here. Data are presented as means ± SDs for each group (n = 3 mice/group from 3 independent experiments). Scale bar, 100 μm. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.
Supplier Page from R&D Systems, a Bio-Techne Brand for Semaphorin 7A Fc Chimera Protein
Available conjugates: Sizes Available: 50 ug