Journal: Cell death and differentiation

Article Title: Regulation of Semaphorin3A in the process of cutaneous wound healing.

doi: 10.1038/s41418-022-00981-6

Figure Lengend Snippet: Fig. 1 Sema3A-overexpressing adenovirus plasmids contributed to decreased keratinocyte migration and proliferation. A Transfection efficiency was confirmed by qRT-PCR analysis in Hacat and NHEK cells. Bars indicate the mean fold changes ± SEM relative to the control; n = 4. B The effect of Sema3A adenovirus plasmids on the proliferation potential of Hacat cells was analysed by CCK-8 and Colony formation experiments. Data are shown as means ± SEM; n = 4. C The effect of Ad-Sema3A on the proliferation potential of NHEK cells was analysed by CCK-8. Data are shown as means ± SEM; n = 4. D Wound healing assays were performed in Ad-Sema3A or si-Sema3A-transfected Hacat and NHEK cells. The percentage of wound closure is displayed as the mean ± SEM; n = 3. E Transwell assays showed that transfection with adenovirus Seam3A restrained the migratory ability, while Sema3A inhibition reversed this effect. Bars indicate the mean fold changes ± SEM relative to the corresponding control; n = 3. F Angiogenesis in HUVEC during the incubation with supernatant gathered from Sema3A- or si- Sema3A-transfected keratinocytes. G Western blotting analysis of EMT markers in Hacat cells transfected with Ad-Sema3A, si-Sema3A and the relative control. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: Nontargeting scramble, Sema3A siRNA and NRP1 siRNA were purchased from Santa Cruz Biotechnology.

Techniques: Migration, Transfection, Quantitative RT-PCR, Control, CCK-8 Assay, Inhibition, Incubation, Western Blot

Journal: Cell death and differentiation

Article Title: Regulation of Semaphorin3A in the process of cutaneous wound healing.

doi: 10.1038/s41418-022-00981-6

Figure Lengend Snippet: Fig. 2 Sema3A transfection suppressed TGF-β1-induced keratinocyte migration in a NRP1-dependent manner. A Western blotting analysis of Sema3A and EMT markers after exposure to escalated concentrations of TGF-β1 in Hacat and NHEK cells. B Wound healing experiment of incubation with TGF-β1. Data are shown as means ± SEM; n = 3. C Sema3A adenovirus plasmids were transfected into keratinocytes in the absence or presence of TGF-β1. The expression of EMT markers and the phosphorylation of Smad2/3 were shown by western blotting. Wound healing (D) and Transwell (E) assays in transfected Ad-Sema3A keratinocytes in the absence or presence of TGF-β1. The percentage of wound closure is displayed as the mean ± SEM; n = 3. For the transwell assays, bars indicate the mean fold changes ± SEM relative to the corresponding control; n = 3. F EMT-related proteins were determined in Ad-Sema3A ± si-NRP1-transfected cells with or without TGF-β1. Phenotypic alterations were verified by wound healing (G) and transwell (H) assays. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: Nontargeting scramble, Sema3A siRNA and NRP1 siRNA were purchased from Santa Cruz Biotechnology.

Techniques: Transfection, Migration, Western Blot, Incubation, Expressing, Phospho-proteomics, Control

Journal: Cell death and differentiation

Article Title: Regulation of Semaphorin3A in the process of cutaneous wound healing.

doi: 10.1038/s41418-022-00981-6

Figure Lengend Snippet: Fig. 3 Ad-Sema3A transfection suppressed activation of EGFR/ERK axis. A Ad-Seam3A plasmids were transfected into NHEK cells for 48 h. Then, recombinant EGF protein was added to the transfected cells for 15 min. Sema3A, p-EGFR and p-ERK were analysed by western blot. B Wound healing and Transwell assays in transfected Sema3A plasmids in the absence or presence of EGF. C Recombinant EGF protein was incubated in the Ad-Sema3A- or NC-transfected cells for 2 days, and immunoblotting analysis is displayed. D Transfection of short peptides interfering with Sema3A function in keratinocytes, treatment with the EGFR signal inhibitor erlotinib, and testing of the protein expression of EMT markers. E Cells treated with si-Sema3A ± erlotinib were plated in the chamber, and the migration capacity was assessed. Bars indicate the fold changes ± SEM relative to the negative control. F The ERK-specific inhibitor U0126 was introduced into NHEKs. Western blot analysis showed that U0126 attenuated the EMT process mediated by TGF-β1 and that Sema3A deficiency enhanced the protein expression of mesenchymal markers triggered by U0126. G qRT-PCR analysis was performed to confirm the expression level of transcriptional factors including Sema3A, NRP1, GATA-1, CEBPA, XBP1, TP53, CEBPB and TCF4 in Hacat cells. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: Nontargeting scramble, Sema3A siRNA and NRP1 siRNA were purchased from Santa Cruz Biotechnology.

Techniques: Transfection, Activation Assay, Recombinant, Western Blot, Incubation, Expressing, Migration, Negative Control, Quantitative RT-PCR

Journal: Cell death and differentiation

Article Title: Regulation of Semaphorin3A in the process of cutaneous wound healing.

doi: 10.1038/s41418-022-00981-6

Figure Lengend Snippet: Fig. 4 Loss of Sema3A delayed cutaneous wound healing in vivo. A Schematic representation of the wound-healing studies performed in K14-CreTM+;Sema3AL/L and K14-CreTM-;Sema3AL/L mice. B qRT-PCR analysis of epidermal Sema3A mRNA after tamoxifen induction. GAPDH served as control. Bars indicate the mean fold changes ± SEM relative to the control (K14-CreTM-;Sema3AL/L group); n = 3. C Quantification of the wound closure area at different time points after wounding in the exp (K14-CreTM+;Sema3AL/L) and con (K14-CreTM-;Sema3AL/L) groups. Data are shown as means ± SEM; n = 6. D Representative macroscopic illustration of wound healing in exp and con animals at Days 0, 4, 7 and 14. E H&E-stained sections of wounds used for morphometric analysis of the percentage of wound closure (length of newly formed epithelium (NFE)/length of NFE + length of gap between edges of wound epithelium (red dotted line) × 100) and re-epithelialization (length of NFE). White asterisk (*) indicates the proliferative connective tissue in the control group. Scale bar = 200 µm. F Quantification of the percentage of Sema3A/ZEB2 + area of the epithelial and granulation tissue at different time points. G Quantification of the percentage of wound re- epithelialization at Days 7 and 14 after wounding in K14-CreTM+;Sema3AL/L and K14-CreTM-;Sema3AL/L wounds. Data are shown as means ± SEM; n = 6. H Comparison of the healing times (scab falling off) in the days after wounding. Data are shown as means ± SEM; n = 6. I Comparison of connective tissue in control and Sema3A cKO mice. Data are shown as means ± SEM; n = 6. J Thickness of crust after injury. Bars indicate the mean fold changes relative to con (K14-CreTM-;Sema3AL/L) ± SEM; n = 6. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: Nontargeting scramble, Sema3A siRNA and NRP1 siRNA were purchased from Santa Cruz Biotechnology.

Techniques: In Vivo, Quantitative RT-PCR, Control, Staining, Comparison

Journal: Cell death and differentiation

Article Title: Regulation of Semaphorin3A in the process of cutaneous wound healing.

doi: 10.1038/s41418-022-00981-6

Figure Lengend Snippet: Fig. 5 Enhancement of keratinocyte migration upon Rb-Sema3A treatment. Epithelial cells extracted from the injured (Day 4) margin of K14-CreTM+;Sema3AL/L and K14-CreTM-;Sema3AL/L mice were cultured, and the proliferation and migration capacity was determined by CCK-8 (A), wound healing (B) and Transwell assays (C) in vitro. D Morphology of keratinocytes. Immunofluorescence staining of F-actin in cells from the K14-CreTM+;Sema3AL/L (exp) and K14-CreTM-;Sema3AL/L (con) groups. White arrows point to spindle morphological alterations in the control group. E Western blot analysis of EMT markers by Day 4 after injury. Lysates were extracted from the injured margins of sema3A cKO or control mice. F The effect of Rb-Sema3A on the proliferation potential of Hacat cells was analysed by CCK-8. Data are shown as means ± SEM; n = 4. G Wound healing assays were performed in Rb-Sema3A-incubated Hacat and NHEK cells. The percentage of wound closure is displayed as the mean ± SEM; n = 3. H Transwell assays showed that incubated with recombinant Seam3A enhanced the migratory ability of NHEK cells. Bars indicate the mean fold changes ± SEM relative to the corresponding control; n = 3. I Two 8-mm excisional wounds were created on the back of each 7–8-week-old BALB/c nude mouse. Sema3A-transfected Hacat cells or recombinant Sema3A proteins as well as the relative control were injected subcutaneously in the margin (2 mm from the incision) of the wound in nude mice. Photographs were taken at Days 0, 4, 7, 14 and 21. The thickness of the connective tissues (J), time of scar falling (K) and area of wound are displayed (L, M). *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: Nontargeting scramble, Sema3A siRNA and NRP1 siRNA were purchased from Santa Cruz Biotechnology.

Techniques: Migration, Cell Culture, CCK-8 Assay, In Vitro, Staining, Control, Western Blot, Incubation, Recombinant, Transfection, Injection

Journal: Cell death and differentiation

Article Title: Regulation of Semaphorin3A in the process of cutaneous wound healing.

doi: 10.1038/s41418-022-00981-6

Figure Lengend Snippet: Fig. 6 Successive recruitment of Sema3A and NRP1 proteins during the process of wound healing. A Schematic representation of the wound-healing studies performed in K14-CreTM+;Sema3AL/L and K14-CreTM-;Sema3AL/L mice. B qRT-PCR analysis of epidermal Sema3A mRNA after tamoxifen induction. GAPDH served as control. Bars indicate the mean fold changes ± SEM relative to the control (K14-CreTM-;Sema3AL/L

Article Snippet: Nontargeting scramble, Sema3A siRNA and NRP1 siRNA were purchased from Santa Cruz Biotechnology.

Techniques: Quantitative RT-PCR, Control

Journal: Cell death and differentiation

Article Title: Regulation of Semaphorin3A in the process of cutaneous wound healing.

doi: 10.1038/s41418-022-00981-6

Figure Lengend Snippet: Fig. 7 Interaction between NRP1 and EGFR signaling in keratinocytes. A Detection of the EGFR-ERK pathway and EMT inducers by western blotting in Hacat cells incubated with recombinant Sema3A and EGF for 48 h. B Protein levels of NRP1, EGFR, p-EGFR, ERK and p-ERK in cells transfected with si-NRP1. C Localization of NRP1 and EGFR proteins in Hacat cells. Scale bar = 20 µm. D Co-IP experiment between NRP1 and EGFR. IP: NRP1. WB: EGFR. E EGFR- and NRP1-overexpressing Hacat cells were treated with cycloheximide (CHX) for the indicated time periods to inhibit de novo protein synthesis. As a control, MG132 was added to block the catalytic activity. F si-NRP1 and EGFR plasmids were cotransfected into NHEK cells for 48 h. Protein levels of NRP1, EGFR, p-EGFR, ERK and p-ERK were determined by western blot. G NHEK cells were transfected with si-NRP1 plasmids for 2 days before EGF (50 ng/ml) stimulation. Then the IF analysis of EGFR or NRP1 was showed. Scale bar = 20 µm. H NHEKs were stimulated with EGF (100 ng/ml) for the indicated periods of time. IFs were subsequently conducted in the resulting cells to monitor EGFR and NRP1 localization/expression. Scale bar = 20 µm.

Article Snippet: Nontargeting scramble, Sema3A siRNA and NRP1 siRNA were purchased from Santa Cruz Biotechnology.

Techniques: Western Blot, Incubation, Recombinant, Transfection, Co-Immunoprecipitation Assay, Control, Blocking Assay, Activity Assay, Expressing

Journal: Cell death and differentiation

Article Title: Regulation of Semaphorin3A in the process of cutaneous wound healing.

doi: 10.1038/s41418-022-00981-6

Figure Lengend Snippet: Fig. 8 Synergetic effect of Rb-EGF and Rb-Sema3A through EGFR/ERK signaling regulated by NRP1. A NHEKs were serum starved and subsequently stimulated with Rb-Sema3A for 5 min to 1 h and subjected to IF analyses. B Combined treatment with Rb-EGF and Rb-Sema3A was utilized in keratinocytes at the indicated time points. EGFR and NRP1 localization/expression was shown by IF staining. Wound healing assays (C) and Transwell assays (D) were performed in Rb-Sema3A-,Rb-EGF and EGFR plasmids incubated in Hacat cells after transfected with si-NRP1 or negative control. The percentage of wound closure is displayed as the mean ± SEM; n = 3. Bars indicate the mean fold changes ± SEM relative to the corresponding control; n = 3. E Western blot analysis of EGFR-ERK pathway after treatment at the indicated time points. F Immunofluorescence in Hacat cells. si-NRP1 or si-NC plasmids were transfected in EGFR-overexpressing cells. Recombinant EGF and Sema3A cytokines were added in cells to test the process of NRP1 and EGFR activation and degradation. Scale bar = 20 µm. G A schematic of the proposed mechanism. **P < 0.01; ***P < 0.001.

Article Snippet: Nontargeting scramble, Sema3A siRNA and NRP1 siRNA were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Staining, Incubation, Transfection, Negative Control, Control, Western Blot, Recombinant, Activation Assay

Journal: Cell

Article Title: A mast cell receptor mediates post-stroke brain inflammation via a dural-brain axis

doi: 10.1016/j.cell.2025.06.045

Figure Lengend Snippet: (A) Representative flow cytometry gating of WT/ Mrgprb2 −/− brains 48h post-tMCAO. left , Live cells are gated using an immune marker (CD45) and myeloid marker (CD11b). right , Myeloid cells are gated using Ly6G and Ly6C to delineate CD45-high neutrophils and monocytes/macrophages, and CD45-low microglia. (B-D) (B) Absolute count of neutrophils, (C) monocytes/macrophages, and (D) CD11b-positive microglia in contralateral/stroke brain hemispheres of WT/ Mrgprb2 −/− mice 48h post-tMCAO (WT n =18, Mrgprb2 −/− n =16). (E) Representative immunofluorescence images of WT (left)/ Mrgprb2 −/− (right) right brain hemispheres 48h post-tMCAO. top row, CD45-positive immune cells. middle row, GFAP-positive activated astrocytes. Dotted white line separates infarcted tissue without GFAP and live, injured brain tissue with GFAP. bottom row, Merged channels with DAPI. Scale bar=50 μm. (F), left, CCL2 and right, CCL3 protein expression measured by ELISA in contralateral/stroke brain hemispheres of WT/ Mrgprb2 −/− mice (CCL2: WT n =6, Mrgprb2 −/− n =6, CCL3: WT n =5, Mrgprb2 −/− n =5). (G-H) (G) IL-6 and (H) neutrophil elastase protein expression measured by ELISA in contralateral/stroke brain hemispheres of WT/ Mrgprb2 −/− mice (G, WT n =5, Mrgprb2 −/− n =5, H, WT n =7, Mrgprb2 −/− n =7). (I) Representative immunofluorescence image of Mrgprb2 −/− mouse meninges 8 weeks after engraftment with Mrgprb2-tdT mast cells. left column, tdT denotes engrafted cells. middle column, Avidin denotes all mast cells. right column, Merged channels with DAPI. Scale bar=50 μm. (J) Mast cell count in meninges of Mrgprb2 −/− mice engrafted with saline, WT, or Mrgprb2 −/− mast cells, determined by Avidin-positive cells in the dura, using a 0.408mm 2 viewing frame (WT saline n =4, WT engraftment n =5, Mrgprb2 −/− saline n =4, Mrgprb2 −/− engraftment n =4). (K) Absolute count of neutrophils and monocytes/macrophages in contralateral/stroke brain hemispheres of WT/ Mrgprb2 −/− mast cell engrafted mice (WT n =16, Mrgprb2 −/− n =18). (L). left, Representative T2-weighted MRIs, and right , stroke volume of WT/ Mrgprb2 −/− MC-engrafted Mrgprb2 −/− mice 48h post-tMCAO (WT n =9, Mrgprb2 −/− n =8). Statistical analyses: Kruskal-Wallis test (B and G), two-way ANOVA with Sidak’s multiple comparisons test (C-D, F, H, and J-K), and two-sided Student’s t-test (L). Statistical test for C, D, and K was performed on log-transformed data to adjust for non-normality. Bar graphs indicate mean ± SEM. * P < 0.05, ** P < 0.01, **** P < 0.0001.

Article Snippet: Mouse Semaphorin 3A ELISA Kit , Cusabio , Cat#: CSB-EL020980MO.

Techniques: Flow Cytometry, Marker, Immunofluorescence, Expressing, Enzyme-linked Immunosorbent Assay, Avidin-Biotin Assay, Cell Counting, Saline, Transformation Assay

Journal: Oncotarget

Article Title: Axon guidance molecule semaphorin3A is a novel tumor suppressor in head and neck squamous cell carcinoma

doi: 10.18632/oncotarget.6831

Figure Lengend Snippet: ( A ) Immunohistochemistry (IHC) staining for SEMA3A in normal oral epithelium. SEMA3A is highly expressed in normal oral epithelium. ( B ) IHC staining for SEMA3A in HNSCC specimens. SEMA3A is absent or reduced from HNSCC specimens. ( C ) Specimens without incubation with polyclonal antibody served as a negative control. (Scale bar: 100 μm). ( D ) Kaplan-Meier overall survival (OS) curves for 100 patients with HNSCC, according to SEMA3A expression level.

Article Snippet: 48 hours later, culture medium were collected to measure the amount of SEMA3A using the human SEMA3A ELISA kit (Elabscience Biotechnology), according to the manufacturer's instructions.

Techniques: Immunohistochemistry, Incubation, Negative Control, Expressing

Journal: Oncotarget

Article Title: Axon guidance molecule semaphorin3A is a novel tumor suppressor in head and neck squamous cell carcinoma

doi: 10.18632/oncotarget.6831

Figure Lengend Snippet: Correlation of SEMA3A expression and the clinical-pathological parameters of HNSCC specimens

Article Snippet: 48 hours later, culture medium were collected to measure the amount of SEMA3A using the human SEMA3A ELISA kit (Elabscience Biotechnology), according to the manufacturer's instructions.

Techniques: Expressing

Journal: Oncotarget

Article Title: Axon guidance molecule semaphorin3A is a novel tumor suppressor in head and neck squamous cell carcinoma

doi: 10.18632/oncotarget.6831

Figure Lengend Snippet: Univariate and multivariate cox regression analysis of clinical characteristics and SEMA3A expression

Article Snippet: 48 hours later, culture medium were collected to measure the amount of SEMA3A using the human SEMA3A ELISA kit (Elabscience Biotechnology), according to the manufacturer's instructions.

Techniques: Expressing

Journal: Oncotarget

Article Title: Axon guidance molecule semaphorin3A is a novel tumor suppressor in head and neck squamous cell carcinoma

doi: 10.18632/oncotarget.6831

Figure Lengend Snippet: ( A ) SEMA3A expression in HNSCC cell lines HN4, HN6, HN13, CAL27, SCC9 and SCC25 assessed by Western blot analysis (up), and quantification of the protein expression (down). ( B ) SEMA3A expression of CAL27, HN6 and SCC25 cells infected with SEMA3A adenovirus on protein (up) and gene (down) levels, detected by Western blot and real-time RT-PCR. ( C ) Images of colonies of SEMA3A-transduced cells (Ad-SEMA3A-CAL27, Ad-SEMA3A-HN6) and control vector (Ad-Con-CAL27, Ad-Con-HN6) stained with crystal violet (up), and the quantification of cell colonies (down). Each data point represents the mean ± SD of data from 3 independent trials. (** P < 0.01) ( D ) Transfection efficiency of SCC9, HN4 and HN13 cells determined by Western blot (up) and real-time RT-PCR (down), after 48 hours’ SEMA3A-siRNA transfection. ( E ) Images of colonies of SEMA3A-siRNA-transfected cells (Si-SEMA3A-SCC9, Si-SEMA3A-HN4) and negative control (Scramble) cells (Si-Scramble-SCC9, Si-Scramble-HN4) stained with crystal violet (left), and the quantification of cell colonies (right). ( F ) Graphs of growth curves of Ad-SEMA3A-cells (grey lines), Ad-Con-cells (black lines) and control cells (dotted lines), as CCK-8 assays carried out over 5 days. ( G, H ) Images of protein expression of CDKs (2, 4, 6), cyclins (E1, D1, D3) and inhibitors of CDKs (P21, P27) in SEMA3A-over-expressed cells (G) or SEMA3A-siRNA-transfected cells (H). ( I ) Flow cytometric analysis of cell-cycle changes in Ad-SEMA3A/Con-CAL27 and Ad-SEMA3A/Con-HN6 cells.

Article Snippet: 48 hours later, culture medium were collected to measure the amount of SEMA3A using the human SEMA3A ELISA kit (Elabscience Biotechnology), according to the manufacturer's instructions.

Techniques: Expressing, Western Blot, Infection, Quantitative RT-PCR, Control, Plasmid Preparation, Staining, Transfection, Negative Control, CCK-8 Assay

Journal: Oncotarget

Article Title: Axon guidance molecule semaphorin3A is a novel tumor suppressor in head and neck squamous cell carcinoma

doi: 10.18632/oncotarget.6831

Figure Lengend Snippet: ( A ) A gain of apoptosis-like phenotype of SEMA3A-transfected cells on 48 and 96 hours after transfection. Red arrowheads indicated apoptotic cells. Scale bar: 100 μm. ( B ) Flow cytometric analysis of apoptosis in Ad-SEMA3A/Con-CAL27/HN6. Cells were stained with Annexin V-allophycocyanin (APC) and 7-aminoactinomycin D (7-AAD), followed by FACS (fluorescence-activated cell sorting) analysis (up). Apoptosis was determined by FACS analysis (early apoptotic death cells in lower right plot quadrants and late apoptotic death cells in upper right plot quadrants) and plotted (down). As showed, apoptosis rate in Ad-SEMA3A cells was significantly higher than controls (** P < 0.01, t -test). ( C ) Protein changes of caspase (caspase-3, -5, -7) induced by SEMA3A overexpression and depletion were measured by western blotting. Representative images of WB are shown. ( D ) CAL27 cells were treated with rhSEMA3A protein for 48 hours of different concentrations. As the concentration increased, more cells adopted an apoptosis-like phenotype. The remaining cells were counted and plotted. Error bars represent the SD (standard deviation) from three separate experiments. *or**, statistically significant difference compared with the control at p < 0.05 or p < 0.01, respectively.

Article Snippet: 48 hours later, culture medium were collected to measure the amount of SEMA3A using the human SEMA3A ELISA kit (Elabscience Biotechnology), according to the manufacturer's instructions.

Techniques: Transfection, Staining, Fluorescence, FACS, Over Expression, Western Blot, Concentration Assay, Standard Deviation, Control

Journal: Oncotarget

Article Title: Axon guidance molecule semaphorin3A is a novel tumor suppressor in head and neck squamous cell carcinoma

doi: 10.18632/oncotarget.6831

Figure Lengend Snippet: ( A ) HNSCC xenograft tumor models were established. Images of mice with xenograft tumors were showed 7 weeks after adenovirus injection. Red arrow heads indicated xenograft tumors. ( B ) An illustration of scheme of CAL27 cells injection and adenovirus administration. ( C ) Tumor growth curves of nude mice in Ad-control group (Group 1, n = 5) and Ad-SEMA3A group (Group 2, n = 5). ( D ) Subcutaneous tumors isolated from nude mice 7 weeks after adenovirus injection.

Article Snippet: 48 hours later, culture medium were collected to measure the amount of SEMA3A using the human SEMA3A ELISA kit (Elabscience Biotechnology), according to the manufacturer's instructions.

Techniques: Injection, Control, Isolation

Journal: Oncotarget

Article Title: Axon guidance molecule semaphorin3A is a novel tumor suppressor in head and neck squamous cell carcinoma

doi: 10.18632/oncotarget.6831

Figure Lengend Snippet: ( A ) Cellular morphological changes were shown 48 hours after SEMA3A depletion and rhSEMA3A protein (100 ng/ml) treatment. ( B ) Western blot analysis were used to assess the expression of epithelial (E-cadherin and β-catenin) and mesenchymal (N-cadherin and Vimentin) markers inAd-SEMA3A/Con cells (CAL27, SCC25) and Si-SEMA3A/Scramble cells (HN4, SCC9). ( C ) The differences in the migration ability between the Ad-SEMA3A-CAL27/HN6 and Ad-Con-CAL27/HN6 cells, were measured using the wound-healing assay. ( D ) A transwell assay was employed to analyze the cell invasion ability.

Article Snippet: 48 hours later, culture medium were collected to measure the amount of SEMA3A using the human SEMA3A ELISA kit (Elabscience Biotechnology), according to the manufacturer's instructions.

Techniques: Western Blot, Expressing, Migration, Wound Healing Assay, Transwell Assay

Journal: Oncotarget

Article Title: Axon guidance molecule semaphorin3A is a novel tumor suppressor in head and neck squamous cell carcinoma

doi: 10.18632/oncotarget.6831

Figure Lengend Snippet: ( A ) Real time RT-PCR was used to detect the expression of classic EMT-inducers Zeb (Zeb1, Zeb2) and Snail (SNAI1, SNAI2) protein in Ad-SEMA3A/Con-CAL27 cells. ( B ) Western blotting was used to detect the expression of SNAI2 protein in Ad-SEMA3A/Con cells (CAL27, SCC25) and Si-SEMA3A/Con cells (HN4, SCC9). ( C ) Western blotting was performed to assess the expression of NF-κB pathway-related proteins in cytoplasmic and nuclear extracts from Ad-SEMA3A/Con-HN6 and Si-SEMA3A/Scramble-SCC9 cells. β-actin and histone H1 were employed as the positive controls for cytoplasmic and nuclear proteins, respectively. Semi-quantitative analysis of changes in protein expression as determined by scanning of the immunoreactive bands. ( D ) A glycosylated form of NRP1 was detected by Western blot analysis.

Article Snippet: 48 hours later, culture medium were collected to measure the amount of SEMA3A using the human SEMA3A ELISA kit (Elabscience Biotechnology), according to the manufacturer's instructions.

Techniques: Quantitative RT-PCR, Expressing, Western Blot

Journal: Oncotarget

Article Title: Axon guidance molecule semaphorin3A is a novel tumor suppressor in head and neck squamous cell carcinoma

doi: 10.18632/oncotarget.6831

Figure Lengend Snippet: Expression of SEMA3A in normal oral epithelium and HNSCC

Article Snippet: 48 hours later, culture medium were collected to measure the amount of SEMA3A using the human SEMA3A ELISA kit (Elabscience Biotechnology), according to the manufacturer's instructions.

Techniques: Expressing

Journal: Scientific reports

Article Title: New functions of Semaphorin 3E and its receptor PlexinD1 during developing and adult hippocampal formation.

doi: 10.1038/s41598-018-19794-0

Figure Lengend Snippet: Figure 1. Low-power photomicrographs illustrating the distribution of Np1 (a,g,m); Np2 (b,h,n); PlnxD1 (c,i,o); Sema3A (d,j,p); Sema3F (e,k,q) and Sema3E (f,l,r) mRNA in the hippocampal formation and adjacent ventrolateral cortex at E14.5 (a–f), E16.5 (g–l) and P0 (m–r). The different regional boundaries are circumscribed by dashed lines. Characteristic corticofugal, entorhino-hippocampal, subiculo-entorhinal and commissural afferent connections are labelled in red, blue, green and pink respectively in the scheme. Note the absence of Np1 labelling in the ventrolateral cortex at E14.5 and E16.5 (arrows in a and g), compared to Np2 (arrows in b and h) and PlnxD1 (c and i). Sema3A levels in the ventrolateral neocortex were intense at E16.5 (arrows in j). In addition, Sema3E levels in lower layers of both the ventrolateral and entorhinal cortices can be seen from E16.5 onwards (arrows in l). Surprisingly, the subicular region was almost absent of semaphorin labelling (asterisk in p–r). Abbreviations: DG = dentate gyrus; EC = entorhinal cortex; H = hippocampus proper; PaS = parasubiculum; PCL = pyramidal cell layer; S = subiculum; VC = ventrolateral neocortex. Scale bars: a = 250 μm pertains to (b–f); g = 250 μm pertains to (h–l) and m = 100 μm pertains to (n–r).

Article Snippet: In addition, some experiments were conducted using commercial full-length cDNA clones Sema3A (MC205153), Sema3E (MC203244) and Sema3F (MC200862) purchased from OriGene Technologies (Rockville, MD, USA).

Techniques:

Journal: Scientific reports

Article Title: New functions of Semaphorin 3E and its receptor PlexinD1 during developing and adult hippocampal formation.

doi: 10.1038/s41598-018-19794-0

Figure Lengend Snippet: Figure 2. Low-power photomicrographs showing examples of the chemorepulsion of hippocampal (a,b,h), entorhinal (c), ventrolateral (d and f) and dorsal (e and g) axons by Sema3A, Sema3F and Sema3E. Explants were obtained at E14.5 (a–d) or E16.5 (e–h), cultured for 2 days in vitro (DIV) and processed for βIII-tubulin (clone TUJ-1) immunostaining. (a) Dotted line defines the boundary between the proximal (P) and the distal (D) quadrant of the explants. Note the strong chemorepulsion of hippocampal axons by Sema3A at E14.5. In addition, Sema3E-mediated chemorepulsion can be seen on hippocampal, entorhinal and ventrolateral cortex at E14.5 (b–d). Examples of Sema3F-mediated chemorepulsion on ventrolateral axons and Sema3E- effects on hippocampal axons can be seen in f and h respectively. This contrasts with what is observed for dorsal neocortical axons (g). (i–m) Representative phalloidin-TRITC stained neuronal processes of cultured entorhinal explants from E16.5, illustrating semaphorin-mediated growth cone collapse. (i) Normal growth cones, with lamellipodia and filopodia (arrows) from entorhinal explants cultured with SEAP medium. (j–l) Examples of collapsed growth cones (asterisks) after incubation with Sema3A (j), Sema3E (k) and Sema3F (l). (m) Histogram illustrating percentages of collapsed growth cones per explant after the incubation of entorhinal explants with secreted semaphorins. Results represent the mean ± S.E.M. of three separate experiments. Asterisks indicate statistical differences between groups and controls. ***P ≤ 0.001; ANOVA Bonferroni post hoc test. Abbreviations: CA = CA1-3 hippocampal regions; EC = entorhinal cortex; DC = dorsal neocortex; VC = ventrolateral neocortex. Scale bars: a = 150 μm pertains to (b–h); i = 20 μm pertains to (j–l).

Article Snippet: In addition, some experiments were conducted using commercial full-length cDNA clones Sema3A (MC205153), Sema3E (MC203244) and Sema3F (MC200862) purchased from OriGene Technologies (Rockville, MD, USA).

Techniques: Cell Culture, In Vitro, Immunostaining, Staining, Incubation

Journal: Cell & Bioscience

Article Title: An increase in Semaphorin 3A biases the axonal direction and induces an aberrant dendritic arborization in an in vitro model of human neural progenitor differentiation

doi: 10.1186/s13578-022-00916-1

Figure Lengend Snippet: Sema 3A activates the microglia proinflammatory M1 phenotype 48 h after transfection. A Representative immunofluorescence pictures of the percentage of microglia cells transfected with Sema 3A-GFP (Sema 3A) or GFP empty vector (GFP). As control, we used non-transfected microglia cells (Ctrl). Scale bar: 55 µm. 10 × objective. B ELISA assay on media from Sema 3A, GFP and Ctrl microglia 48 h after transfection. Sema 3A levels were expressed as ng/ml and normalized on the number of alive cells (mean ± SEM from 10 different fields) for each experimental point. Data are the mean ± SEM of three independent experiments performed in triplicate. ****P < 0.0001 vs Sema 3A media. C Extent of cell survival obtained by counting the number of DAPI positive nuclei before and after Sema 3A transfection as well as in GFP and Ctrl microglia. Data are the mean ± SEM of three independent experiments in quadruplicate. One-way ANOVA followed by Tukey’s test for multiple comparisons. ****P < 0.0001 vs Sema 3A. D Quantitative analysis of TMEM 119, Iba1, CD86, CD68, and E iNOS and TNFα positive cells. MFI was performed on the entire slide using Zeiss Celldiscoverer7. Values are normalized on the number of DAPI positive cells for each slide and expressed as % of Ctrl. Data are the mean ± SEM of three independent experiments performed in quadruplicate. One-way ANOVA followed by Tukey’s test for multiple comparisons. *P < 0.05; **P < 0.01; ***P < 0.001. Representative images of iNOS (red) and TNFα (red) and Sema 3A or GFP (green) staining are reported in F . Scale bar: 10 µm. 63 × objective

Article Snippet: Sema 3A protein levels in media from microglia overexpressing Sema 3A (Sema 3A media) or GFP (GFP media) or non-transfected (Ctrl media) were assessed using Elisa kit (ELISA, Cusabio, Houston, TX, USA #CSB-E15913h), according to the manufacturer instructions.

Techniques: Transfection, Immunofluorescence, Plasmid Preparation, Control, Enzyme-linked Immunosorbent Assay, Staining

Journal: Experimental eye research

Article Title: Expression of axon guidance ligands and their receptors in the cornea and trigeminal ganglia and their recovery after corneal epithelium injury

doi: 10.1016/j.exer.2022.109054

Figure Lengend Snippet: Antibodies used for immunofluorescence staining of trigeminal ganglia and cornea sections.

Article Snippet: Semaphorin3A , Rabbit , 1:300 , all , Proteintech 21925 , Donkey anti rabbit Cy3 , 1:400 , Jackson Immunoresearch 711-025-152.

Techniques: Immunofluorescence, Staining

Journal: Experimental eye research

Article Title: Expression of axon guidance ligands and their receptors in the cornea and trigeminal ganglia and their recovery after corneal epithelium injury

doi: 10.1016/j.exer.2022.109054

Figure Lengend Snippet: Basal and post-injury expression of axon guidance proteins in mouse cornea. Mice were subjected to corneal epithelium debridement, and the expression of these proteins was followed up in a time-dependent manner. All of these proteins are well-expressed in the uninjured cornea. After debridement, they quickly recovered, and their expression was visible from day 3 to day 14 post-injury. When compared to uninjured corneas, EphrinB1 was highly expressed at all post-injury days, while EphrinB2 and Sema3A where highly expressed from day 7 to day 14. The expression of Sema3F, Netrin-1 and Netrin-4 at day 14 was similar to the basal conditions. Quantification of immunostaining described in Methods is shown in the graphs. Representative images of the central debrided cornea, n = 3 mice per time point. Scale bar = 50 μm.

Article Snippet: Semaphorin3A , Rabbit , 1:300 , all , Proteintech 21925 , Donkey anti rabbit Cy3 , 1:400 , Jackson Immunoresearch 711-025-152.

Techniques: Expressing, Immunostaining

Journal: Experimental eye research

Article Title: Expression of axon guidance ligands and their receptors in the cornea and trigeminal ganglia and their recovery after corneal epithelium injury

doi: 10.1016/j.exer.2022.109054

Figure Lengend Snippet: Variable gene expression recovery of axon guidance proteins in injured mouse cornea. Mouse corneal epithelium was harvested from uninjured mice corneas or animals that received cornea epithelium debridement. Gene expression was analyzed as described in Materials and Methods. We found that 3 days after injury, only the expression of EphrinB2 was significantly upregulated, while all other axon guidance ligands were downregulated. At later time points, Sema3A, EphrinB1, and EphrinB2 were upregulated as well, while Sema3F, Netrin-1 and Netrin-4 were downregulated. In general, all of these proteins trend to normalize their expression to the basal level, and only EphrinB1 was still showing high level of expression after 4 weeks post-injury. N = 5 mice per time point. * = p < 0.05.

Article Snippet: Semaphorin3A , Rabbit , 1:300 , all , Proteintech 21925 , Donkey anti rabbit Cy3 , 1:400 , Jackson Immunoresearch 711-025-152.

Techniques: Gene Expression, Expressing

Journal: Experimental eye research

Article Title: Expression of axon guidance ligands and their receptors in the cornea and trigeminal ganglia and their recovery after corneal epithelium injury

doi: 10.1016/j.exer.2022.109054

Figure Lengend Snippet: Axon guidance proteins accelerated corneal epithelial wound closure. Human corneal limbal epithelial (HCLE) cells were grown to confluency on a 24-well plate and then a scratch was performed using a scratcher tip as described in Methods. Cells were then treated with recombinant Sema3A, EphrinB2, Netrin-4 or left untreated as control. Cells were immediately placed in a chamber incubator on a microscope and images were collected every 20 min for 15 h and analyzed using Fiji software. All the axon guidance proteins accelerated the wound closure when compared to control. (A) Representative images of scratch area. Sema3A (B) and EphrinB2 (C) induced faster healing with complete closure at 8 h, while Netrin-4 (D) did at 10 h, still faster than control. Each treatment was performed in triplicate and 3 images were obtained per treatment. Experiments were performed three times. Scale bar = 400 μm. * = p < 0.05; ** = p < 0.01. Values are mean ± S.E.M.

Article Snippet: Semaphorin3A , Rabbit , 1:300 , all , Proteintech 21925 , Donkey anti rabbit Cy3 , 1:400 , Jackson Immunoresearch 711-025-152.

Techniques: Recombinant, Control, Microscopy, Software

Journal: Journal of Biological Chemistry

Article Title: Phosphorylation of Cyclin-dependent Kinase 5 (Cdk5) at Tyr-15 Is Inhibited by Cdk5 Activators and Does Not Contribute to the Activation of Cdk5

doi: 10.1074/jbc.m113.501148

Figure Lengend Snippet: FIGURE 6. Tyr-15 phosphorylation of Cdk5 in neurons treated with Sema3A, Ephrin-A1, or BDNF. A, immunoblots of neuronal lysates with anti- phospho-Tyr-15 Cdk5 (pY15). Mouse cortical neurons prepared from wild- type (/) or Cdk5 knockout (/) mouse brains were immunoblotted with antibodies to Cdk5 C8, anti-phospho-Tyr-15 of Cdk5 (sc) and (ab), and actin. B, an immunoblot of anti-Cdk5 C8 immunoprecipitates with anti-phospho- Tyr-15 (sc). Cdk5 was immunoprecipitated (IP) from the extract (Ext) of mouse brains at embryonic day18.5, which was treated with Na3VO4 (V) and ATP. The supernatant (sup) or immunoprecipitate (ppt) was immunoblotted with anti- Cdk5 C8 or control IgG, followed by immunoblotting with anti-phospho- Tyr-15 Cdk5 (sc) (top panel) or anti-Cdk5 C8 (bottom panel). C, phospho-Cdk5 at Tyr-15 did not coimmunoprecipitate with anti-p35. Cultured neuronal lysates were immunoprecipitated with anti-Cdk5 C8 or anti-p35 C19, and Cdk5 phosphorylated at Tyr-15 was detected with anti-phospho-Tyr-15 Cdk5 (sc). D, Tyr-15 phosphorylation in neurons treated with Sema3A, Ephrin-A1, or BDNF.MousecorticalneuronsweretreatedwithSema3A,Ephrin-A1(Eph-A1), BDNF, or 100 M pervanadate (PV) for the indicated times. Cell lysates were immunoblotted with antibodies to Cdk5 (third panel), p35 (fourth panel), anti- phospho-SFK (fifth panel), or phospho-Thr-202/Tyr-204 of ERK (p-MAPK, sixth panel). Phosphorylation of Tyr-15 on Cdk5 was detected with anti-phospho- Tyr-15 Cdk5 (sc) (first panel) after immunoprecipitation with anti-Cdk5 C8 (second panel). Lane 10 is a molecular weight (MW) marker. The asterisk indi- cates carbonic anhydrase at 32.2 kDa. Lanes 11 and 12 are Cdk5 coexpressed with or without caFyn in HEK293 cells for reference. E, the effect of Sema3A on neurite retraction. Neurites of mouse cortical neurons expressing EGFP were observed by time-lapse imaging at intervals of 5 min after addition of Sema3A. Scale bar 5 m. F, kinase activity of Cdk5 after Sema3A treatment. Cdk5 was prepared from cultured neurons treated with Sema3A for 30 min by immunoprecipitation, and its kinase activity was measured by phosphoryla- tion of histone H1. Data are mean S.E. (n 3). ns, not significant; Student’s t test. Ctrl, control. G, the protein ratio of p35 and Cdk5 in COS-7 cells and mouse brain extract. FLAG-Cdk5 and FLAG-p35 were transfected into COS-7 cells using 1 g of plasmid for each of Cdk5 and p35, the same as in Fig. 2. Immunoblotting was performed by adjusting Cdk5 approximately (bottom panel). FLAG-p35 expressed in COS-7 cells is indicated by an arrowhead, and p35 in the brain extract (BrE) is indicated by an arrow (top panel). H, the effect of p35 at the low expression levels (similar to the brain extract) on Tyr-15 phosphorylation of Cdk5. COS-7 cells were transfected with FLAG-Cdk5 and caFyn with or without FLAG-p35 as shown in Fig. 2A, except for the reduced plasmid amount of p35 (1/40). Phosphorylation of Tyr-15 in Cdk5 was detected by immunoblotting with antibodies to phospho-Tyr-15 of Cdk5 (ab). Blotting of Cdk5, p35, Fyn, and actin is also shown.

Article Snippet: Semaphorin 3A (Sema3A) and Ephrin-A1 were from R&D Systems (Pune, Maharashtra, India).

Techniques: Phospho-proteomics, Western Blot, Knock-Out, Immunoprecipitation, Control, Cell Culture, Molecular Weight, Marker, Expressing, Imaging, Activity Assay, Transfection, Plasmid Preparation