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Fig. 1 <t>Sema3A-overexpressing</t> 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 <t>Sema3A</t> 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.

Sema3a Sirna, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Regulation of Semaphorin3A in the process of cutaneous wound healing."

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

Journal: Cell death and differentiation

doi: 10.1038/s41418-022-00981-6

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.

Figure Legend 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.

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

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.

Figure Legend 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.

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

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.

Figure Legend 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.

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

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.

Figure Legend 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.

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

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.

Figure Legend 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.

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

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

Figure Legend 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

Techniques Used: Quantitative RT-PCR, Control

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.

Figure Legend 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.

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

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.

Figure Legend 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.

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

Transfection:

Article Title: Axon guidance molecule semaphorin3A is a novel tumor suppressor in head and neck squamous cell carcinoma
Article Snippet: .. Cells at 30–40% confluence were transfected with 10 μM SEMA3A-siRNA or Control-siRNA (Santa Cruz Biotechnology, Inc.) according to the manufacturer's instructions. ..

Article Title: Axon guidance molecule semaphorin3A is a novel tumor suppressor in head and neck squamous cell carcinoma.
Article Snippet: .. Cells at 30–40% confluence were transfected with 10 μM SEMA3A-siRNA or Control-siRNA (Santa Cruz Biotechnology, Inc.) according to the manufacturer’s instructions. ..

Control:

Image Search Results

(A) Schematic illustration of the synthesis of AuNP@siRNA-Sema3A using electrostatic interactions. TEM images of (B) AuNP, (C) AuNP-PLL, and (D) AuNP@siRNA-Sema3A. (E) Changes in the surface charge of each AuNP according to the step-by-step coating of PLL and siRNA-Sema3A. (F) Confocal micrograph of NSCs treated with Cy5.5-labeled AuNP@siRNA-Sema3A (left) and details (right) of the internalization of Cy5.5-labeled AuNP@siRNA-Sema3A into the cells

Journal: Biomaterials Research

Article Title: Axon guidance gene-targeted siRNA delivery system improves neural stem cell transplantation therapy after spinal cord injury

doi: 10.1186/s40824-023-00434-2

Figure Lengend Snippet: (A) Schematic illustration of the synthesis of AuNP@siRNA-Sema3A using electrostatic interactions. TEM images of (B) AuNP, (C) AuNP-PLL, and (D) AuNP@siRNA-Sema3A. (E) Changes in the surface charge of each AuNP according to the step-by-step coating of PLL and siRNA-Sema3A. (F) Confocal micrograph of NSCs treated with Cy5.5-labeled AuNP@siRNA-Sema3A (left) and details (right) of the internalization of Cy5.5-labeled AuNP@siRNA-Sema3A into the cells

Article Snippet: Sema3A siRNA (siRNA-Sema3A) was obtained from Bioneer (Seoul, South Korea).

Techniques: Labeling

Differentiating NSCs express Sema3A, and AuNP@siRNA-Sema3A suppresses Sema3A expression (A-D) Representative immunofluorescence images of Sema3A (anti-Sema3A, Green), neurons (anti-Tuj1, Red), and nuclei (DAPI, Blue) of the (A) non-treated, (B) AuNP, (C) Hydrogel, and (D) AuNP@siRNA-Sema3A groups. (E) Quantitative analyses of the fluorescence intensity for Sema3A. (F-I) Representative immunofluorescence images for Tuj1-labelled axon growth of the (F) non-treated group, (G) AuNP, (H) Hydrogel, and (I) AuNP@siRNA-Sema3A groups. (J) Quantitative analyses of Tuj1-labelled total axon growth in ROIs. Results are the mean ± standard error of the mean (SEM); *** p < 0.001. one-way ANOVA with Tukey post-hoc test

Journal: Biomaterials Research

Article Title: Axon guidance gene-targeted siRNA delivery system improves neural stem cell transplantation therapy after spinal cord injury

doi: 10.1186/s40824-023-00434-2

Figure Lengend Snippet: Differentiating NSCs express Sema3A, and AuNP@siRNA-Sema3A suppresses Sema3A expression (A-D) Representative immunofluorescence images of Sema3A (anti-Sema3A, Green), neurons (anti-Tuj1, Red), and nuclei (DAPI, Blue) of the (A) non-treated, (B) AuNP, (C) Hydrogel, and (D) AuNP@siRNA-Sema3A groups. (E) Quantitative analyses of the fluorescence intensity for Sema3A. (F-I) Representative immunofluorescence images for Tuj1-labelled axon growth of the (F) non-treated group, (G) AuNP, (H) Hydrogel, and (I) AuNP@siRNA-Sema3A groups. (J) Quantitative analyses of Tuj1-labelled total axon growth in ROIs. Results are the mean ± standard error of the mean (SEM); *** p < 0.001. one-way ANOVA with Tukey post-hoc test

Article Snippet: Sema3A siRNA (siRNA-Sema3A) was obtained from Bioneer (Seoul, South Korea).

Techniques: Expressing, Immunofluorescence, Fluorescence

Transplantation of NSCs induces Sema3A in the grafted site and AuNP@siRNA-Sema3A successfully inhibits the expression of Sema3A after SCI. Immunofluorescence analysis in composite tiled scans of transverse sections stained for glial scarring (anti-GFAP, Red) and Sema3A (anti-Sema3A, white). Astrocytic glial scar (AS) around the lesion center (LC). (A) Representative merged image of GFAP and Sema3A in the SCI group (top). Higher magnification in the LC (left) and in the AS (right) of the SCI group. (B) Representative merged image of GFAP and Sema3A in the SCI + NSC group (top). Higher magnification in the LC (left) and in the AS (right) of the SCI + NSC group. (C) Representative merged image of GFAP and Sema3A in the SCI + NSC + siRNA-Sema3A group (top). Higher magnification in the LC (left) and in the AS (right) of the SCI + NSC + siRNA-Sema3A group. (D-E) Quantitative analyses of the fluorescence intensity for Sema3A in the (D) LC and (E) AS. Results are the mean ± SEM; *** p < 0.001. one-way ANOVA with Tukey post hoc test

Journal: Biomaterials Research

Article Title: Axon guidance gene-targeted siRNA delivery system improves neural stem cell transplantation therapy after spinal cord injury

doi: 10.1186/s40824-023-00434-2

Figure Lengend Snippet: Transplantation of NSCs induces Sema3A in the grafted site and AuNP@siRNA-Sema3A successfully inhibits the expression of Sema3A after SCI. Immunofluorescence analysis in composite tiled scans of transverse sections stained for glial scarring (anti-GFAP, Red) and Sema3A (anti-Sema3A, white). Astrocytic glial scar (AS) around the lesion center (LC). (A) Representative merged image of GFAP and Sema3A in the SCI group (top). Higher magnification in the LC (left) and in the AS (right) of the SCI group. (B) Representative merged image of GFAP and Sema3A in the SCI + NSC group (top). Higher magnification in the LC (left) and in the AS (right) of the SCI + NSC group. (C) Representative merged image of GFAP and Sema3A in the SCI + NSC + siRNA-Sema3A group (top). Higher magnification in the LC (left) and in the AS (right) of the SCI + NSC + siRNA-Sema3A group. (D-E) Quantitative analyses of the fluorescence intensity for Sema3A in the (D) LC and (E) AS. Results are the mean ± SEM; *** p < 0.001. one-way ANOVA with Tukey post hoc test

Article Snippet: Sema3A siRNA (siRNA-Sema3A) was obtained from Bioneer (Seoul, South Korea).

Techniques: Transplantation Assay, Expressing, Immunofluorescence, Staining, Fluorescence

Knockdown of Sema3A increases survival of grafted NSCs and induces neural differentiation after transplantation in the injured spinal cord. Immunofluorescence analysis in composite tiled scans of transverse sections stained for GFP-labeled NSCs (anti-GFP, Green) and glial scar (anti-GFAP, Red). (A, B) Representative merged image for GFP and GFAP in the (A) SCI + NSC group, and (B) SCI + NSC + siRNA-Sema3A group. (C, D) Higher magnification in the LC (boxed area) (C) from A and (D) from B. (E, F) Higher magnification of the AS (boxed area) (E) from A and (F) from B

Journal: Biomaterials Research

Article Title: Axon guidance gene-targeted siRNA delivery system improves neural stem cell transplantation therapy after spinal cord injury

doi: 10.1186/s40824-023-00434-2

Figure Lengend Snippet: Knockdown of Sema3A increases survival of grafted NSCs and induces neural differentiation after transplantation in the injured spinal cord. Immunofluorescence analysis in composite tiled scans of transverse sections stained for GFP-labeled NSCs (anti-GFP, Green) and glial scar (anti-GFAP, Red). (A, B) Representative merged image for GFP and GFAP in the (A) SCI + NSC group, and (B) SCI + NSC + siRNA-Sema3A group. (C, D) Higher magnification in the LC (boxed area) (C) from A and (D) from B. (E, F) Higher magnification of the AS (boxed area) (E) from A and (F) from B

Article Snippet: Sema3A siRNA (siRNA-Sema3A) was obtained from Bioneer (Seoul, South Korea).

Techniques: Transplantation Assay, Immunofluorescence, Staining, Labeling

Knockdown of Sema3A can improve synaptic connectivity between grafted NSCs and host neurons after SCI. (A-G) Immunofluorescence analysis in merged images of transverse sections stained for GFP-labeled NSCs (anti-GFP, Green), neurons (anti-Tuj1, Red), and nuclei (DAPI, Blue) or synapse (anti-Homer, White). (A) Representative composite tiled scan merged images of GFP and GFAP in the SCI + NSC + siRNA-Sema3A group. (B-D) Higher magnification for GFP, Tuj1, and DAPI (B) ahead of the proximal border (PB), (C) at the grafted site, and (D) in the distal border (DB) from A. (E-G) Higher magnification for GFP, Tuj1, and Homer in the boxed area (E) from B, (F) from C, and (G) from D

Journal: Biomaterials Research

Article Title: Axon guidance gene-targeted siRNA delivery system improves neural stem cell transplantation therapy after spinal cord injury

doi: 10.1186/s40824-023-00434-2

Figure Lengend Snippet: Knockdown of Sema3A can improve synaptic connectivity between grafted NSCs and host neurons after SCI. (A-G) Immunofluorescence analysis in merged images of transverse sections stained for GFP-labeled NSCs (anti-GFP, Green), neurons (anti-Tuj1, Red), and nuclei (DAPI, Blue) or synapse (anti-Homer, White). (A) Representative composite tiled scan merged images of GFP and GFAP in the SCI + NSC + siRNA-Sema3A group. (B-D) Higher magnification for GFP, Tuj1, and DAPI (B) ahead of the proximal border (PB), (C) at the grafted site, and (D) in the distal border (DB) from A. (E-G) Higher magnification for GFP, Tuj1, and Homer in the boxed area (E) from B, (F) from C, and (G) from D

Article Snippet: Sema3A siRNA (siRNA-Sema3A) was obtained from Bioneer (Seoul, South Korea).

Techniques: Immunofluorescence, Staining, Labeling

Knockdown of Sema3A enhances laminin expression and improves motor function recovery after SCI (A) Immunofluorescence analysis in composite tiled scans of transverse sections stained for AS (anti-GFAP, Red) and laminin (anti-laminin, White). (B) Quantitative analyses of the laminin percentage in the LC. Results are the mean ± SEM; *** p < 0.001. one-way ANOVA with Tukey post hoc test. (C) Comparison of BBB locomotor scores in the SCI, SCI + NSC, and SCI + NSC + siRNA-Sema3A group. # denotes a significant difference between the SCI group and the SCI + NSC group. * denotes a significant difference between the SCI group and the SCI + NSC + siRNA-Sema3A group. $ denotes a significant difference between the SCI + NSC group and the SCI + NSC + siRNA-Sema3A group. Results are the mean ± SD; # p < 0.05, $ p < 0.05, $$ p < 0.01, $$$ p < 0.001, and *** p < 0.001. Unpaired two-tailed Student’s t-tests

Journal: Biomaterials Research

Article Title: Axon guidance gene-targeted siRNA delivery system improves neural stem cell transplantation therapy after spinal cord injury

doi: 10.1186/s40824-023-00434-2

Figure Lengend Snippet: Knockdown of Sema3A enhances laminin expression and improves motor function recovery after SCI (A) Immunofluorescence analysis in composite tiled scans of transverse sections stained for AS (anti-GFAP, Red) and laminin (anti-laminin, White). (B) Quantitative analyses of the laminin percentage in the LC. Results are the mean ± SEM; *** p < 0.001. one-way ANOVA with Tukey post hoc test. (C) Comparison of BBB locomotor scores in the SCI, SCI + NSC, and SCI + NSC + siRNA-Sema3A group. # denotes a significant difference between the SCI group and the SCI + NSC group. * denotes a significant difference between the SCI group and the SCI + NSC + siRNA-Sema3A group. $ denotes a significant difference between the SCI + NSC group and the SCI + NSC + siRNA-Sema3A group. Results are the mean ± SD; # p < 0.05, $ p < 0.05, $$ p < 0.01, $$$ p < 0.001, and *** p < 0.001. Unpaired two-tailed Student’s t-tests

Article Snippet: Sema3A siRNA (siRNA-Sema3A) was obtained from Bioneer (Seoul, South Korea).

Techniques: Expressing, Immunofluorescence, Staining, Comparison, Two Tailed Test

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 death and differentiation

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

doi: 10.1038/s41418-022-00981-6

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

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

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

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

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

MRNA Sema3A was highly expressed in IS and correlated with cell viability and apoptosis. ( A ) The mRNA level of Sema3A in N2a cells climbed up with time after 6h, 12h, 24h of OGD/R treatment. * P <0.05 vs control. ( B ) Successful downregulation of Sema3A by si- Sema3A . * P <0.05 vs control. ( C ) Sema3A downregulation led to higher viability of OGD/R cells. * P <0.05 vs control, # P <0.05 vs siRNA-NC. ( D , E ) OGD/R cells have higher apoptosis rate compared with control groups while Sema3A downregulation is correlated with lower apoptosis level among OGD/R cells. * P <0.05 vs control, # P <0.05 vs siRNA-NC. ( F ) OGD/R cells tend to have higher level of c-caspase-3 as well as p-JNK and p-p38 in comparison with control group. Downregulation of Sema3A could counteract with this trend in OGD/R cells partly. * P <0.05 vs control, # P <0.05 vs siRNA-NC.

Journal: Aging (Albany NY)

Article Title: LncRNA MEG3 targeting miR-424-5p via MAPK signaling pathway mediates neuronal apoptosis in ischemic stroke

doi: 10.18632/aging.102790

Figure Lengend Snippet: MRNA Sema3A was highly expressed in IS and correlated with cell viability and apoptosis. ( A ) The mRNA level of Sema3A in N2a cells climbed up with time after 6h, 12h, 24h of OGD/R treatment. * P <0.05 vs control. ( B ) Successful downregulation of Sema3A by si- Sema3A . * P <0.05 vs control. ( C ) Sema3A downregulation led to higher viability of OGD/R cells. * P <0.05 vs control, # P <0.05 vs siRNA-NC. ( D , E ) OGD/R cells have higher apoptosis rate compared with control groups while Sema3A downregulation is correlated with lower apoptosis level among OGD/R cells. * P <0.05 vs control, # P <0.05 vs siRNA-NC. ( F ) OGD/R cells tend to have higher level of c-caspase-3 as well as p-JNK and p-p38 in comparison with control group. Downregulation of Sema3A could counteract with this trend in OGD/R cells partly. * P <0.05 vs control, # P <0.05 vs siRNA-NC.

Article Snippet: MiR-424-5p mimics, inhibitor, MEG3 siRNAs and Sema3A siRNAs (listed in ) were designed and purchased from Sigma Aldrich (St. Louis, MO, USA).

Techniques:

MiR-424-5p targeted with Sema3A and was lowly expressed in IS. ( A ) MiR-424-5p was among 11 miRNAs which both targeted Sema3A and related to MAPK signaling pathway. ( B ) The relative miR-424-5p expression declined with time of 6h, 12h, 24h of OGD/R treatment. * P <0.05 vs control. ( C , D ) The target relationship between Sema3A and miR-424-5p was predicted by Targetscan and verified by dual luciferase assay. * P <0.05 vs control. ( E , F ) Upregulation of miR-424-5p by miR-424-5p mimics led to lower Sema3A expression, while downregulation of miR-424-5p led to higher Sema3A expression. * P <0.05 vs control.

Journal: Aging (Albany NY)

Article Title: LncRNA MEG3 targeting miR-424-5p via MAPK signaling pathway mediates neuronal apoptosis in ischemic stroke

doi: 10.18632/aging.102790

Figure Lengend Snippet: MiR-424-5p targeted with Sema3A and was lowly expressed in IS. ( A ) MiR-424-5p was among 11 miRNAs which both targeted Sema3A and related to MAPK signaling pathway. ( B ) The relative miR-424-5p expression declined with time of 6h, 12h, 24h of OGD/R treatment. * P <0.05 vs control. ( C , D ) The target relationship between Sema3A and miR-424-5p was predicted by Targetscan and verified by dual luciferase assay. * P <0.05 vs control. ( E , F ) Upregulation of miR-424-5p by miR-424-5p mimics led to lower Sema3A expression, while downregulation of miR-424-5p led to higher Sema3A expression. * P <0.05 vs control.

Article Snippet: MiR-424-5p mimics, inhibitor, MEG3 siRNAs and Sema3A siRNAs (listed in ) were designed and purchased from Sigma Aldrich (St. Louis, MO, USA).

Techniques: Expressing, Luciferase

MEG3 was a highly expressed lncRNA in IS that bonded with miR-424-5p. ( A ) The heatmap from the microarray analysis of GSE22255 showed the significantly differentially expressed lncRNAs including MEG3 in the IS group. ( B ) MEG3 bound to miR-424-5p and was related to IS. ( C ) MEG3 expression was positively correlated with OGD/R intervention time. * P <0.05 vs control. ( D ) The target relationship between MEG3 and miR-424-5p was predicted and proved. * P <0.05 vs control ( E ) The effect of downregulation MEG3 by si-MEG3 was proved significant. * P <0.05 vs control. ( F ) MEG3 knockdown correlated with higher miR-424-5p expression. * P <0.05 vs control. ( G ) MEG3 knockdown correlated with lower Sema3A expression. * P <0.05 vs control.

Journal: Aging (Albany NY)

Article Title: LncRNA MEG3 targeting miR-424-5p via MAPK signaling pathway mediates neuronal apoptosis in ischemic stroke

doi: 10.18632/aging.102790

Figure Lengend Snippet: MEG3 was a highly expressed lncRNA in IS that bonded with miR-424-5p. ( A ) The heatmap from the microarray analysis of GSE22255 showed the significantly differentially expressed lncRNAs including MEG3 in the IS group. ( B ) MEG3 bound to miR-424-5p and was related to IS. ( C ) MEG3 expression was positively correlated with OGD/R intervention time. * P <0.05 vs control. ( D ) The target relationship between MEG3 and miR-424-5p was predicted and proved. * P <0.05 vs control ( E ) The effect of downregulation MEG3 by si-MEG3 was proved significant. * P <0.05 vs control. ( F ) MEG3 knockdown correlated with higher miR-424-5p expression. * P <0.05 vs control. ( G ) MEG3 knockdown correlated with lower Sema3A expression. * P <0.05 vs control.

Article Snippet: MiR-424-5p mimics, inhibitor, MEG3 siRNAs and Sema3A siRNAs (listed in ) were designed and purchased from Sigma Aldrich (St. Louis, MO, USA).

Techniques: Microarray, Expressing

The MEG3/miR-424-5p/ Sema3A axis affected cell viability and apoptosis in OGD cells via the MAPK pathway. ( A ) OGD/R cells have lower cell viability compared with control group. Among OGD/R cells, MEG3 downregulation led to higher cell viability, which could be offset by a simultaneous overexpression of Sema3A ; miR-424-5p downregulation by its inhibitor led to a lower cell viability compared among OGD/R cells, which could be neutralized by a simultaneous downregulation of Sema3A . * P <0.05 vs control, # P <0.05 vs OGD/R NC, $ P <0.05 vs si-MEG3, & P <0.05 vs inhibitor ( B , C ) OGD/R cells tend to have fairly high apoptosis rate compared to control group. Among OGD/R cells, MEG3 downregulation led to lower cell apoptosis level, which could be neutralized by a simultaneous overexpression of Sema3A ; miR-424-5p downregulation by its inhibitor mediated more cell apoptosis, which could be neutralized by a simultaneous downregulation of Sema3A . * P <0.05 vs control, # P <0.05 vs OGD/R NC, $ P <0.05 vs si-MEG3, & P <0.05 vs inhibitor. ( D ) OGD/R cells tend to have higher cleaved caspase-3, p-JNK and p-p38 expression in comparison with control. Among OGD/R cells, the low expression of MEG3 led to a rise in expression of cleaved caspase-3, p-JNK and p-p38, miR-424-5p inhibitor led to drop of expression of cleaved caspase-3, p-JNK and p-p38. Sema3A overexpression could reverse the rise of si-MEG3, while si- Sema3A could reverse the drop of miR-424-5p inhibitor. * P <0.05 vs control, # P <0.05 vs OGD/R NC, $ P <0.05 vs si-MEG3, & P <0.05 vs inhibitor.

Journal: Aging (Albany NY)

Article Title: LncRNA MEG3 targeting miR-424-5p via MAPK signaling pathway mediates neuronal apoptosis in ischemic stroke

doi: 10.18632/aging.102790

Figure Lengend Snippet: The MEG3/miR-424-5p/ Sema3A axis affected cell viability and apoptosis in OGD cells via the MAPK pathway. ( A ) OGD/R cells have lower cell viability compared with control group. Among OGD/R cells, MEG3 downregulation led to higher cell viability, which could be offset by a simultaneous overexpression of Sema3A ; miR-424-5p downregulation by its inhibitor led to a lower cell viability compared among OGD/R cells, which could be neutralized by a simultaneous downregulation of Sema3A . * P <0.05 vs control, # P <0.05 vs OGD/R NC, $ P <0.05 vs si-MEG3, & P <0.05 vs inhibitor ( B , C ) OGD/R cells tend to have fairly high apoptosis rate compared to control group. Among OGD/R cells, MEG3 downregulation led to lower cell apoptosis level, which could be neutralized by a simultaneous overexpression of Sema3A ; miR-424-5p downregulation by its inhibitor mediated more cell apoptosis, which could be neutralized by a simultaneous downregulation of Sema3A . * P <0.05 vs control, # P <0.05 vs OGD/R NC, $ P <0.05 vs si-MEG3, & P <0.05 vs inhibitor. ( D ) OGD/R cells tend to have higher cleaved caspase-3, p-JNK and p-p38 expression in comparison with control. Among OGD/R cells, the low expression of MEG3 led to a rise in expression of cleaved caspase-3, p-JNK and p-p38, miR-424-5p inhibitor led to drop of expression of cleaved caspase-3, p-JNK and p-p38. Sema3A overexpression could reverse the rise of si-MEG3, while si- Sema3A could reverse the drop of miR-424-5p inhibitor. * P <0.05 vs control, # P <0.05 vs OGD/R NC, $ P <0.05 vs si-MEG3, & P <0.05 vs inhibitor.

Article Snippet: MiR-424-5p mimics, inhibitor, MEG3 siRNAs and Sema3A siRNAs (listed in ) were designed and purchased from Sigma Aldrich (St. Louis, MO, USA).

Techniques: Over Expression, Expressing

MEG3 expression accelerated the process of IS by promoting Sema3A expression in vivo . ( A ) Expression level of MEG3 increased gradually at 4h, 8h, 24h point in the process of ischemia reperfusion (I/R stands for ischemic reperfusion). * P <0.05 vs sham. ( B ) Downregulation of MEG3 expression by si-MEG3 was proved significant. Simultaneous upregulation of Sema3A had little influence on the MEG3 expression. * P <0.05 vs control. ( C ) Sema3A expression was suppressed by si-MEG3, and the decrease was neutralized by manual Sema3A overexpression. * P <0.05 vs control, # P <0.05 vs si-MEG3. ( D ) Representative image of brain slices of three groups of rats. Normal tissues were a pink or red color, whereas the ischemic tissues were white. MCAO rats tend to have a bigger ischemic area in contrast with Sham group. Among MCAO rats, si-MEG3 group with MEG3 down-regulated have smaller ischemic area while it could be reversed by overexpression of Sema3A . ( E ) Quantified average percentage of the infarction area in the whole brain, the percentage was higher in MCAO rats compared with sham group. Among MCAO rats, the infarct volume decreased in si-MEG3 group, while simultaneous overexpression of Sema3A weakened the decrease. * P <0.05 vs sham, # P <0.05 vs MCAO, $ P <0.05 vs MCAO+si-MEG3. ( F ) Neurological score rose in MCAO rats compared with sham rats. Among MCAO rats, si-MEG3 group had lower neurological score than MCAO control, while simultaneous overexpression of Sema3A neutralized the decrease of score. * P <0.05 vs sham, # P <0.05 vs MCAO, $ P <0.05 vs MCAO+si-MEG3. ( G ) Expression of cleaved caspase-3, p-JNK and p-p38 increased significantly in MCAO group compared with control group. In si-MEG3 group, expression of cell apoptosis markers cleaved caspase-3 and phosphorylated levels of JNK and p38 decreased. Overexpression of Sema3A reversed the change. * P <0.05 vs sham, # P <0.05 vs MCAO, $ P <0.05 vs MCAO+si-MEG3.

Journal: Aging (Albany NY)

Article Title: LncRNA MEG3 targeting miR-424-5p via MAPK signaling pathway mediates neuronal apoptosis in ischemic stroke

doi: 10.18632/aging.102790

Figure Lengend Snippet: MEG3 expression accelerated the process of IS by promoting Sema3A expression in vivo . ( A ) Expression level of MEG3 increased gradually at 4h, 8h, 24h point in the process of ischemia reperfusion (I/R stands for ischemic reperfusion). * P <0.05 vs sham. ( B ) Downregulation of MEG3 expression by si-MEG3 was proved significant. Simultaneous upregulation of Sema3A had little influence on the MEG3 expression. * P <0.05 vs control. ( C ) Sema3A expression was suppressed by si-MEG3, and the decrease was neutralized by manual Sema3A overexpression. * P <0.05 vs control, # P <0.05 vs si-MEG3. ( D ) Representative image of brain slices of three groups of rats. Normal tissues were a pink or red color, whereas the ischemic tissues were white. MCAO rats tend to have a bigger ischemic area in contrast with Sham group. Among MCAO rats, si-MEG3 group with MEG3 down-regulated have smaller ischemic area while it could be reversed by overexpression of Sema3A . ( E ) Quantified average percentage of the infarction area in the whole brain, the percentage was higher in MCAO rats compared with sham group. Among MCAO rats, the infarct volume decreased in si-MEG3 group, while simultaneous overexpression of Sema3A weakened the decrease. * P <0.05 vs sham, # P <0.05 vs MCAO, $ P <0.05 vs MCAO+si-MEG3. ( F ) Neurological score rose in MCAO rats compared with sham rats. Among MCAO rats, si-MEG3 group had lower neurological score than MCAO control, while simultaneous overexpression of Sema3A neutralized the decrease of score. * P <0.05 vs sham, # P <0.05 vs MCAO, $ P <0.05 vs MCAO+si-MEG3. ( G ) Expression of cleaved caspase-3, p-JNK and p-p38 increased significantly in MCAO group compared with control group. In si-MEG3 group, expression of cell apoptosis markers cleaved caspase-3 and phosphorylated levels of JNK and p38 decreased. Overexpression of Sema3A reversed the change. * P <0.05 vs sham, # P <0.05 vs MCAO, $ P <0.05 vs MCAO+si-MEG3.

Article Snippet: MiR-424-5p mimics, inhibitor, MEG3 siRNAs and Sema3A siRNAs (listed in ) were designed and purchased from Sigma Aldrich (St. Louis, MO, USA).

Techniques: Expressing, In Vivo, Over Expression

Journal: Frontiers in Cellular Neuroscience

Article Title: Semaphorin 3A Contributes to Secondary Blood–Brain Barrier Damage After Traumatic Brain Injury

doi: 10.3389/fncel.2019.00117

Figure Lengend Snippet: Antibodies for Western blot.

Article Snippet: siRNA transfection was carried out in vivo with the method reported previously ( ). siRNA-SEMA3A (0.5 nmol, RiboBio, Guangzhou, China) and siRNA-control (0.5 nmol, RiboBio, Guangzhou, China) were diluted with the same volume of Entranster TM - in vivo transfection reagent (Engreen, Beijing, China).

Techniques: Western Blot, Molecular Weight

TBI altered the expression of SEMA3A and its related receptors, Nrp-1 and plexin-A1. (A) Schematic diagram of the experimental design. (B,C) Quantitative data from western blotting illustrating the time course of SEMA3A following TBI, with an increase on the first day post-TBI and peaked on the third day. (D–F) Quantitative data from western blotting illustrating the time course of the related receptors of SEMA3A following TBI, with an increase on the first day post-TBI and peaked on the third day. (G–L) Double-immunostaining data indicating that SEMA3A was mainly secreted and concentrated at the lesion boundary after TBI. (I) Fluorescence intensity of lesion boundary of sham group. (J) Fluorescence intensity of lesion boundary of TBI group. (K) Fluorescence intensity of contralateral of sham group. (L) Fluorescence intensity of contralateral of TBI group. Scale bar = 50 μm. The data are expressed as the mean ± SEM, and n = 6 for each group. ∗ p < 0.05 vs. sham groups, # p < 0.01 vs. sham group and TBI contralateral group.

Journal: Frontiers in Cellular Neuroscience

Article Title: Semaphorin 3A Contributes to Secondary Blood–Brain Barrier Damage After Traumatic Brain Injury

doi: 10.3389/fncel.2019.00117

Figure Lengend Snippet: TBI altered the expression of SEMA3A and its related receptors, Nrp-1 and plexin-A1. (A) Schematic diagram of the experimental design. (B,C) Quantitative data from western blotting illustrating the time course of SEMA3A following TBI, with an increase on the first day post-TBI and peaked on the third day. (D–F) Quantitative data from western blotting illustrating the time course of the related receptors of SEMA3A following TBI, with an increase on the first day post-TBI and peaked on the third day. (G–L) Double-immunostaining data indicating that SEMA3A was mainly secreted and concentrated at the lesion boundary after TBI. (I) Fluorescence intensity of lesion boundary of sham group. (J) Fluorescence intensity of lesion boundary of TBI group. (K) Fluorescence intensity of contralateral of sham group. (L) Fluorescence intensity of contralateral of TBI group. Scale bar = 50 μm. The data are expressed as the mean ± SEM, and n = 6 for each group. ∗ p < 0.05 vs. sham groups, # p < 0.01 vs. sham group and TBI contralateral group.

Techniques: Expressing, Western Blot, Double Immunostaining, Fluorescence

Influence of SEMA3A on neurological outcomes following TBI. (A) Schematic diagram of the experimental design. (B,C) The expression of SEMA3A was blocked by siRNA-SEMA3A in vivo . (D,E) We used mNSSs to test neurological function (D) and beam balance tests (E) to evaluate motor function on the 1st, 3rd, 7th, and 14th days post-CCI. The downregulation of SEMA3A significantly decreased mNSS scores and reduced the times on the beam balance test on the third day after injury. The data are expressed as the mean ± SEM, and n = 6 for each group. # p < 0.01 vs. control groups, ∗ p < 0.05 TBI+siRNA-SEMA groups vs. siRNA-control groups, ∗∗ p < 0.05 TBI+SEMA3A groups vs. TBI+PBS groups.

Journal: Frontiers in Cellular Neuroscience

Article Title: Semaphorin 3A Contributes to Secondary Blood–Brain Barrier Damage After Traumatic Brain Injury

doi: 10.3389/fncel.2019.00117

Figure Lengend Snippet: Influence of SEMA3A on neurological outcomes following TBI. (A) Schematic diagram of the experimental design. (B,C) The expression of SEMA3A was blocked by siRNA-SEMA3A in vivo . (D,E) We used mNSSs to test neurological function (D) and beam balance tests (E) to evaluate motor function on the 1st, 3rd, 7th, and 14th days post-CCI. The downregulation of SEMA3A significantly decreased mNSS scores and reduced the times on the beam balance test on the third day after injury. The data are expressed as the mean ± SEM, and n = 6 for each group. # p < 0.01 vs. control groups, ∗ p < 0.05 TBI+siRNA-SEMA groups vs. siRNA-control groups, ∗∗ p < 0.05 TBI+SEMA3A groups vs. TBI+PBS groups.

Techniques: Expressing, In Vivo

The impact of SEMA3A on BBB leakage after TBI. (A) The general view of the EB dye extravasation assay in brains of injured mice on the third day after injury. (B) Quantitative analysis of the EB dye extravasation assay presented in (A). (C) The brain water content was measured on the third day after injury. SEMA3A can significantly increase BBB leakage and edema, and all these negative effects post-TBI can be efficiently decreased by blocking the expression of SEMA3A. (D,E) The p-VE-cadherin/VE-cadherin ratio was examined on the third day after injury through western blotting, and the results illustrated that the downregulation of SEMA3A can decrease VE-cadherin serine phosphorylation post-TBI. The data are expressed as the mean ± SEM, and n = 6 for each group. ∗ p < 0.05 TBI+SEMA3A groups vs. TBI+PBS groups, ∗∗ p < 0.05 TBI+siRNA-SEMA groups vs. siRNA-control groups.

Journal: Frontiers in Cellular Neuroscience

Article Title: Semaphorin 3A Contributes to Secondary Blood–Brain Barrier Damage After Traumatic Brain Injury

doi: 10.3389/fncel.2019.00117

Figure Lengend Snippet: The impact of SEMA3A on BBB leakage after TBI. (A) The general view of the EB dye extravasation assay in brains of injured mice on the third day after injury. (B) Quantitative analysis of the EB dye extravasation assay presented in (A). (C) The brain water content was measured on the third day after injury. SEMA3A can significantly increase BBB leakage and edema, and all these negative effects post-TBI can be efficiently decreased by blocking the expression of SEMA3A. (D,E) The p-VE-cadherin/VE-cadherin ratio was examined on the third day after injury through western blotting, and the results illustrated that the downregulation of SEMA3A can decrease VE-cadherin serine phosphorylation post-TBI. The data are expressed as the mean ± SEM, and n = 6 for each group. ∗ p < 0.05 TBI+SEMA3A groups vs. TBI+PBS groups, ∗∗ p < 0.05 TBI+siRNA-SEMA groups vs. siRNA-control groups.

Techniques: Blocking Assay, Expressing, Western Blot

OGD induced changes in SEMA3A expression in endothelial cells and contributes to BBB leakage induced by OGD. (A–E) To investigate the causes of the alterations of SEMA3A post-injury, we treated endothelial cells (bEnd.3) with OGD for 4 h and tested protein expression levels with western blotting. The results indicated that compared with those in the control group, which was cultured under normoxic conditions, the expression levels of SEMA3A (A,B) , Nrp-1 and plexin-A1 (C–E) in the OGD group significantly increased. (F,G) The expression of SEMA3A was blocked by siRNA-SEMA3A in vitro . (H) FITC-dextran transport studies showed that the amount of FITC-dextran that was transported through the transwell into the lower chamber dramatically increased in the OGD group and the SEMA3A group. This amount decreased when SEMA3A was blocked. (I) We treated bEnd.3 cells with the SEMA3A protein and observed the morphological changes by HPICM. The results illustrated that SEMA3A can efficiently increase defects of the BBB. (J) Observations of the integrity of the barrier by HPICM indicated that the destruction of the barrier that is induced by OGD is efficiently impaired by the downregulation of SEMA3A expression. (K,L) Testing of tight junction changes post-OGD with western blotting revealed that SEMA3A contributes to VE-cadherin serine phosphorylation. The data are expressed as the mean ± SEM, and n = 6 for each group. ∗ p < 0.01 vs. control group, & p < 0.01 vs. control group, ∗∗ p < 0.05 vs. control groups, ∗& p < 0.05 vs. OGD+PBS group, #∗ p < 0.01 vs. siRNA-control group, # p < 0.05 vs. OGD+PBS group.

Journal: Frontiers in Cellular Neuroscience

Article Title: Semaphorin 3A Contributes to Secondary Blood–Brain Barrier Damage After Traumatic Brain Injury

doi: 10.3389/fncel.2019.00117

Figure Lengend Snippet: OGD induced changes in SEMA3A expression in endothelial cells and contributes to BBB leakage induced by OGD. (A–E) To investigate the causes of the alterations of SEMA3A post-injury, we treated endothelial cells (bEnd.3) with OGD for 4 h and tested protein expression levels with western blotting. The results indicated that compared with those in the control group, which was cultured under normoxic conditions, the expression levels of SEMA3A (A,B) , Nrp-1 and plexin-A1 (C–E) in the OGD group significantly increased. (F,G) The expression of SEMA3A was blocked by siRNA-SEMA3A in vitro . (H) FITC-dextran transport studies showed that the amount of FITC-dextran that was transported through the transwell into the lower chamber dramatically increased in the OGD group and the SEMA3A group. This amount decreased when SEMA3A was blocked. (I) We treated bEnd.3 cells with the SEMA3A protein and observed the morphological changes by HPICM. The results illustrated that SEMA3A can efficiently increase defects of the BBB. (J) Observations of the integrity of the barrier by HPICM indicated that the destruction of the barrier that is induced by OGD is efficiently impaired by the downregulation of SEMA3A expression. (K,L) Testing of tight junction changes post-OGD with western blotting revealed that SEMA3A contributes to VE-cadherin serine phosphorylation. The data are expressed as the mean ± SEM, and n = 6 for each group. ∗ p < 0.01 vs. control group, & p < 0.01 vs. control group, ∗∗ p < 0.05 vs. control groups, ∗& p < 0.05 vs. OGD+PBS group, #∗ p < 0.01 vs. siRNA-control group, # p < 0.05 vs. OGD+PBS group.

Techniques: Expressing, Western Blot, Cell Culture, In Vitro

miR-30b-5p regulated SEMA3A expression following injury in vivo and in vitro . (A) Compared with those of the sham groups, the RT-PCR results indicated that the expression level of miR-30b-5p in CCI mice decreased and reached its lowest value on the seventh day post-injury. (B) In an in vitro experiment, the expression level of miR-30b-5p decreased in bEnd.3 cells post-OGD. (C,D) In the CCI mouse model, the downregulation of miR-30b-5p increased the expression level of SEMA3A, and the upregulation of miR-30b-5p decreased SEMA3A levels. (E,F) These results were repeated in the OGD model in bEnd.3 cells. (G) Schematic representation of the potential binding sites for miR-30b-5p in the Sema3a 3′UTR. Seed sequences of the WT ( Sema3a WT 3′UTR) and mutant ( Sema3a Mut 3′UTR) luciferase reporters are shown in the binding site. (H) Data from the luciferase assay indicated that miR-30b-5p inhibited the luciferase activity of the WT but not the Mut 3′UTR reporter construct, suggesting that miR-30b-5p could directly target Sema3a and downregulate its expression by binding to the 3′UTR sites. The data are expressed as the mean ± SEM, and n = 6 for each group. ∗ p < 0.05 vs. sham group, ∗∗ p < 0.05 vs. control group, ∗# p < 0.01 vs. control groups, # p < 0.01 vs. control groups, and ∗& p < 0.05 vs. control groups.

Journal: Frontiers in Cellular Neuroscience

Article Title: Semaphorin 3A Contributes to Secondary Blood–Brain Barrier Damage After Traumatic Brain Injury

doi: 10.3389/fncel.2019.00117

Figure Lengend Snippet: miR-30b-5p regulated SEMA3A expression following injury in vivo and in vitro . (A) Compared with those of the sham groups, the RT-PCR results indicated that the expression level of miR-30b-5p in CCI mice decreased and reached its lowest value on the seventh day post-injury. (B) In an in vitro experiment, the expression level of miR-30b-5p decreased in bEnd.3 cells post-OGD. (C,D) In the CCI mouse model, the downregulation of miR-30b-5p increased the expression level of SEMA3A, and the upregulation of miR-30b-5p decreased SEMA3A levels. (E,F) These results were repeated in the OGD model in bEnd.3 cells. (G) Schematic representation of the potential binding sites for miR-30b-5p in the Sema3a 3′UTR. Seed sequences of the WT ( Sema3a WT 3′UTR) and mutant ( Sema3a Mut 3′UTR) luciferase reporters are shown in the binding site. (H) Data from the luciferase assay indicated that miR-30b-5p inhibited the luciferase activity of the WT but not the Mut 3′UTR reporter construct, suggesting that miR-30b-5p could directly target Sema3a and downregulate its expression by binding to the 3′UTR sites. The data are expressed as the mean ± SEM, and n = 6 for each group. ∗ p < 0.05 vs. sham group, ∗∗ p < 0.05 vs. control group, ∗# p < 0.01 vs. control groups, # p < 0.01 vs. control groups, and ∗& p < 0.05 vs. control groups.

Techniques: Expressing, In Vivo, In Vitro, Reverse Transcription Polymerase Chain Reaction, Binding Assay, Mutagenesis, Luciferase, Activity Assay, Construct

The neuroprotective effect of miR-30b-5p in mice following TBI by regulating SEMA3A. (A) Schematic diagram of the experimental design. (B–D) The brain water content (B) , mNSS score (C) and beam balance test (D) were measured on the third day after injury. Upregulation of miR-30b-5p could efficiently decrease brain edema, and improve neurological outcomes post-TBI. The negative effect of recombinant SEMA3A protein also can be improved by upregulation of miR-30b-5p. The data are expressed as the mean ± SEM, and n = 6 for each group, ∗ p < 0.05 vs. control groups, # p < 0.05 vs. TBI+agomir-C+SEMA3A group.

Journal: Frontiers in Cellular Neuroscience

Article Title: Semaphorin 3A Contributes to Secondary Blood–Brain Barrier Damage After Traumatic Brain Injury

doi: 10.3389/fncel.2019.00117

Figure Lengend Snippet: The neuroprotective effect of miR-30b-5p in mice following TBI by regulating SEMA3A. (A) Schematic diagram of the experimental design. (B–D) The brain water content (B) , mNSS score (C) and beam balance test (D) were measured on the third day after injury. Upregulation of miR-30b-5p could efficiently decrease brain edema, and improve neurological outcomes post-TBI. The negative effect of recombinant SEMA3A protein also can be improved by upregulation of miR-30b-5p. The data are expressed as the mean ± SEM, and n = 6 for each group, ∗ p < 0.05 vs. control groups, # p < 0.05 vs. TBI+agomir-C+SEMA3A group.

Techniques: Recombinant

Review

sema3a sirna (Santa Cruz Biotechnology)

Structured Review

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1) Product Images from "Regulation of Semaphorin3A in the process of cutaneous wound healing."

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