Journal: Frontiers in Medicine

Article Title: Adriamycin-induced podocyte injury via the Sema3A/TRPC5/Rac1 pathway

doi: 10.3389/fmed.2024.1381479

Figure Lengend Snippet: Urinary Sema3A and 24 h urinary protein levels in patients with podocytopathies and controls. (A) Urinary Sema3A in patients with podocytopathies and controls. ** p < 0.01. (B) Urinary PCX in patients with podocytopathies and controls. ** p < 0.01. (C) Twenty-four-hour urinary protein in patients with podocytopathies. ** p < 0.01. (D) Scatter plot between urinary Sema3A and PCX ( n = 135).

Article Snippet: Recombinant mouse Semaphorin3A protein from Sino Biological (Beijing, China).

Techniques:

Journal: Frontiers in Medicine

Article Title: Adriamycin-induced podocyte injury via the Sema3A/TRPC5/Rac1 pathway

doi: 10.3389/fmed.2024.1381479

Figure Lengend Snippet: Correlation of urinary protein (g/24 h) levels with urinary Sema3A (ng/ml) and PCX (ng/ml) levels in podocytopathies patients.

Article Snippet: Recombinant mouse Semaphorin3A protein from Sino Biological (Beijing, China).

Techniques:

Journal: Frontiers in Medicine

Article Title: Adriamycin-induced podocyte injury via the Sema3A/TRPC5/Rac1 pathway

doi: 10.3389/fmed.2024.1381479

Figure Lengend Snippet: Adriamycin-induced protein expression in podocytes. (A) Western blot images of Sema3A, Desmin, and podocin protein expression in podocytes. (B–D) Relative quantitative analysis of Sema3A, Desmin, and podocin in Adriamycin-treated podocytes ( n = 3). ** p < 0.01.

Article Snippet: Recombinant mouse Semaphorin3A protein from Sino Biological (Beijing, China).

Techniques: Expressing, Western Blot

Journal: Frontiers in Medicine

Article Title: Adriamycin-induced podocyte injury via the Sema3A/TRPC5/Rac1 pathway

doi: 10.3389/fmed.2024.1381479

Figure Lengend Snippet: Inhibition of Sema3A protects podocytes from Adriamycin-induced injury. (A) Western blotting assay showing the levels of Sema3A in Adriamycin-induced podocytes. (B) Levels of Sema3A in Control, AN, Control siRNA (siRNASema3A-NC), and siRNASema3A, demonstrating a decrease in protein levels with siRNASema3A. (C) Cell viability of podocytes treated with AN+siRNASema3A and AN+siRNASema3A + rhSema3A (200 ng/mL in culture medium). (D) Podocyte migration area decreased in the siRNASema3A group compared with that in the AN and AN+siRNASema3A + rhSema3A groups, as analyzed using scratch wound assay. Scale bar: 100 μm. (E) Quantitative analysis of migration rates in (D) . ** p < 0.01 ( n = 3). (F) Podocyte migration decreased in the siRNASema3A group compared with that in the AN and AN+siRNASema3A + rhSema3A groups, as confirmed using the Transwell assay. Scale bar: 50 μm. (G) Quantitative analysis of migrated cells in (E) . ** p < 0.01 ( n = 3). (H) Determination of apoptotic podocytes using flow cytometry. (I) Relative apoptosis rate decreased following Adriamycin+siRNASema3A and Adriamycin +siRNASema3A + rhSema3A (200 ng/mL in culture medium) treatments compared with that after AN treatment ( n = 3) ** p < 0.01.

Article Snippet: Recombinant mouse Semaphorin3A protein from Sino Biological (Beijing, China).

Techniques: Inhibition, Western Blot, Control, Migration, Scratch Wound Assay Assay, Transwell Assay, Flow Cytometry

Journal: Frontiers in Medicine

Article Title: Adriamycin-induced podocyte injury via the Sema3A/TRPC5/Rac1 pathway

doi: 10.3389/fmed.2024.1381479

Figure Lengend Snippet: Inhibition of Sema3A increases the expression of podocin and decreases that of Desmin in podocytes. (A) Western blotting to assess the levels of Sema3A, podocin, and Desmin proteins in podocytes. (B–D) Relative densities of Sema3A, podocin, and Desmin protein expression normalized to GAPDH ( n = 3) * p < 0.05, ** p < 0.01.

Article Snippet: Recombinant mouse Semaphorin3A protein from Sino Biological (Beijing, China).

Techniques: Inhibition, Expressing, Western Blot

Journal: Frontiers in Medicine

Article Title: Adriamycin-induced podocyte injury via the Sema3A/TRPC5/Rac1 pathway

doi: 10.3389/fmed.2024.1381479

Figure Lengend Snippet: TRPC5 expression increased following Sema3A overexpression in podocytes. (A) Western blotting to assess TRPC5 levels in podocytes. (B) Upregulated TRPC5 expression in response to exogenous Sema3A (200 ng/mL) in the culture medium ( n = 3) * p < 0.05. (C) Podocytes overexpressing Sema3A. (D) Relative mRNA expression of Sema3A and TRPC5 ( n = 3) ** p < 0.01. (E) Relative densities of Sema3A and TRPC5 protein expression normalized to GAPDH ( n = 3) ** p < 0.01.

Article Snippet: Recombinant mouse Semaphorin3A protein from Sino Biological (Beijing, China).

Techniques: Expressing, Over Expression, Western Blot

Journal: Frontiers in Medicine

Article Title: Adriamycin-induced podocyte injury via the Sema3A/TRPC5/Rac1 pathway

doi: 10.3389/fmed.2024.1381479

Figure Lengend Snippet: (A) Western blotting to assess the levels of Desmin, podocin Sema3A, TRPC5, and Rac1 protein in podocytes. (B–F) Relative protein expression of Desmin, podocin, Sema3A, TRPC5, and Rac1 in mice kidney tissue normalized to GAPDH were determined using western blotting. The values are presented as mean ± standard deviation ( n = 3). * p < 0.05, ** p < 0.01.

Article Snippet: Recombinant mouse Semaphorin3A protein from Sino Biological (Beijing, China).

Techniques: Western Blot, Expressing, Standard Deviation

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: Scientific reports

Article Title: Single-nucleus transcriptomic profiling of the diaphragm during mechanical ventilation.

doi: 10.1038/s41598-024-82530-4

Figure Lengend Snippet: Fig. 6. Protein-protein interaction network (PPI network).We constructed nine PPI networks displaying protein-protein interactions among the related genes. The nodes represent proteins, and the edges represent the interaction strength between two proteins. The proteins PFKFB3 (a), PDGFD (b), CXCR2 (c), CCL21 (d), SEMA3A (e), RYR3 (f), MEF2C (g), NEGR1(h) and TBC1D1 (i) which are located at the hub of the interaction network, are responsible for diaphragm fibrosis and atrophy.

Article Snippet: After the semidry blotting procedure (50 min, 90 V), the membrane was incubated for 1 h at room temperature (RT) in 5% BSA blocking solution, followed by overnight incubation on a shaker at 4 °C with primary antibodies against PFKFB3 (bs-3528R, Boster Biological Technology Co., Ltd., Wuhan, China), PDGFD (bs-24572R, Boster), CXCR2 (bs-1629R, Boster), NEGR1 (bs-11095R, Boster), SEMA3A (bs-10468R, Boster), MEF2C (bs-4130R, Boster) and β-actin (66009-1-Ig, Proteintech, Wuhan, China) as a loading control.

Techniques: Construct, Protein-Protein interactions

Journal: Scientific reports

Article Title: Single-nucleus transcriptomic profiling of the diaphragm during mechanical ventilation.

doi: 10.1038/s41598-024-82530-4

Figure Lengend Snippet: Fig. 8. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting. qRT-PCR and Western blotting showing the expression of selected genes and corresponding proteins in the mechanically ventilated diaphragm and control ones. The bar graphs show the quantification of the mRNA expression of Pfkfb3 (a), Pdgfd (b), Cxcr2 (c), Negr1 (d), Sema3a (e), and Mef2c (f) normalized to that of GAPDH. Asterisks indicate significant differences (n = 3 in each group) (*P < 0.05). Western blotting analysis of the protein expression of PFKFB3 (g), PDGFD (h), CXCR2 (i), NEGR1 (j), SEMA3A (k), and MEF2C (l) normalized to that of β-actin as a loading control.

Article Snippet: After the semidry blotting procedure (50 min, 90 V), the membrane was incubated for 1 h at room temperature (RT) in 5% BSA blocking solution, followed by overnight incubation on a shaker at 4 °C with primary antibodies against PFKFB3 (bs-3528R, Boster Biological Technology Co., Ltd., Wuhan, China), PDGFD (bs-24572R, Boster), CXCR2 (bs-1629R, Boster), NEGR1 (bs-11095R, Boster), SEMA3A (bs-10468R, Boster), MEF2C (bs-4130R, Boster) and β-actin (66009-1-Ig, Proteintech, Wuhan, China) as a loading control.

Techniques: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Expressing, Control

Journal: PLoS ONE

Article Title: Semaphorin3A induces nerve regeneration in the adult cornea-a switch from its repulsive role in development

doi: 10.1371/journal.pone.0191962

Figure Lengend Snippet: RT-PCR and qPCR of class 3 Semaphorins was performed in isolated cells and tissues as detailed in Materials and Methods. Immunofluorescence staining was performed in eyes from intact mice (basal control) and mice subjected to corneal epithelium debridement. (A) All class 3 Semaphorins are expressed in the corneal epithelium and TG, with Sema3B and Sema3F showing higher and consistent levels. (B) The gene expression of Sema3A and Sema3F after corneal epithelium debridement significantly changed from their basal level. While Sema3F expression in the corneal epithelium is reduced to 50% after one day post debridement and slowly recovers to normal levels after 2 weeks, Sema3A is sharply upregulated soon after injury and remains elevated over the entire evaluated period. Values represent mean ± SD, experiments were performed in triplicate (for each experiment tissues were collected from n = 4 mice, * indicates p< 0.05 vs day 0). ( C ) Eye crossections stained with anti Sema3A (red) antibody and DAPI (blue, nuclear staining) shows apical expression of Sema3A in basal control. Upon injury there is high expression of Sema3A in the basal layers of the epithelium. Dashed white-square in left image shows the area of analysis. Images are representative of n = 4 mice.

Article Snippet: For immunofluorescence staining, section were fixed in ice cold methanol for 5 min, washed 3 times in PBS, blocked (2% BSA, 2.5% donkey serum in PBS) for 1h at room temperature and incubated overnight with 1:200 dilution of rabbit anti Sema3A antibody (cat # 21925, Proteintech, Rosemont, IL) at 4°C.

Techniques: Reverse Transcription Polymerase Chain Reaction, Isolation, Immunofluorescence, Staining, Control, Gene Expression, Expressing

Journal: PLoS ONE

Article Title: Semaphorin3A induces nerve regeneration in the adult cornea-a switch from its repulsive role in development

doi: 10.1371/journal.pone.0191962

Figure Lengend Snippet: Isolated embryonic and adult DRG were treated with 50 ng/ml NGF to induce neuronal growth and then Sema3A was added to the cultures to test the inhibitory effect using time lapse imaging analysis. ( A) In embryonic DRG, NGF induced a fast growth of neurites that developed long axons with extensive branching and a clear growth cone area (white arrows). (B) After 3 days in culture, Sema3A was added and time-lapse imaged recorded. Addition of Sema3A induced fast growth cone collapse and axonal retraction (white arrows) here shown after 10 h. ( C ) In adult DRG neurons, NGF induced extension and branching of neurites. (D) Addition of Sema3A has no inhibitory effect on the neurite growth and no regression or collapse of axons was observed. Representative images of neurons observed in experiments performed in triplicate, each dish with an average of 200 neurons; all neurons in every dish were evaluated (scale bars A and B = 20 μm, C and D = 100 μm).

Article Snippet: For immunofluorescence staining, section were fixed in ice cold methanol for 5 min, washed 3 times in PBS, blocked (2% BSA, 2.5% donkey serum in PBS) for 1h at room temperature and incubated overnight with 1:200 dilution of rabbit anti Sema3A antibody (cat # 21925, Proteintech, Rosemont, IL) at 4°C.

Techniques: Isolation, Imaging

Journal: PLoS ONE

Article Title: Semaphorin3A induces nerve regeneration in the adult cornea-a switch from its repulsive role in development

doi: 10.1371/journal.pone.0191962

Figure Lengend Snippet: Isolated DRG and TG neurons were incubated with NGF for 3 and 2 days respectively to induce neuronal growth and then treated with different concentrations of Sema3A to determine any significant growth inhibitory effect on these PNS neurons. Neurite growth was evaluated 24 h after addition of Sema3A. (A) We found that about one third of the isolated neurons responded to the NGF treatment and addition of Sema3A produced no inhibitory effects such as the neurons kept growing similarly as compared to control cells treated with NGF alone. ( B ) TG neurons responded to NGF similarly to DRG neurons and addition of Sema3A did not alter the growth of neurites and no axonal retraction was observed. Neuronal growth was similar to controls that were resupplied with NGF over the course of the experiment. Values represent mean ± SEM, experiments were performed in triplicate, each dish had an average of 200 neurons for DRG and 60 neurons for TG; all neurons in every dish were evaluated.

Article Snippet: For immunofluorescence staining, section were fixed in ice cold methanol for 5 min, washed 3 times in PBS, blocked (2% BSA, 2.5% donkey serum in PBS) for 1h at room temperature and incubated overnight with 1:200 dilution of rabbit anti Sema3A antibody (cat # 21925, Proteintech, Rosemont, IL) at 4°C.

Techniques: Isolation, Incubation, Produced, Control

Journal: PLoS ONE

Article Title: Semaphorin3A induces nerve regeneration in the adult cornea-a switch from its repulsive role in development

doi: 10.1371/journal.pone.0191962

Figure Lengend Snippet: Since Sema3A is highly expressed in the cornea upon injury and does not inhibit the NGF induced growth on adult PNS neurons, we evaluated the effects of adding Sema3A alone to cultured neurons and compared to the NGF induced growth. (A) As described above DRG neurons responded well to NGF treatment. Surprisingly, Sema3A by itself is also a potent inducer of neuronal growth and similar neurite extension was observed at doses of 20 ng/ml Sema3A or higher. (B) Similarly, Sema3A is also a potent inducer of neuronal growth on TG neurons, and the number of TG neurons showing neurite growth was similar to that of neurons treated with NGF alone when incubated with Sema3A at 10 ng/ml or higher. (C) Sema3A from different sources, recombinant human or mouse, induced equally strong neuronal growth of TG neurons at equal concentrations. Values represent mean ± SD, experiments were performed in triplicate, each dish had an average of 200 neurons for DRG and 60 neurons for TG, and all neurons in a dish were evaluated. Neurite growth was evaluated at day 4 for DRG neurons and at day 3 for TG neurons. * indicate p<0.05 vs NGF. (R&D = from R&D Systems, m = murine, h = human, SB = Sino Biological Inc.).

Article Snippet: For immunofluorescence staining, section were fixed in ice cold methanol for 5 min, washed 3 times in PBS, blocked (2% BSA, 2.5% donkey serum in PBS) for 1h at room temperature and incubated overnight with 1:200 dilution of rabbit anti Sema3A antibody (cat # 21925, Proteintech, Rosemont, IL) at 4°C.

Techniques: Cell Culture, Incubation, Recombinant

Journal: PLoS ONE

Article Title: Semaphorin3A induces nerve regeneration in the adult cornea-a switch from its repulsive role in development

doi: 10.1371/journal.pone.0191962

Figure Lengend Snippet: The neuronal promoting effects of Sema3A and NGF were compared side by side in adult TG and DRG neurons. Neurons were treated either with growth medium alone as negative control or with 50ng/ml NGF or Sema3A. (A) The length of the neurites was quantified after 48 and 72 h post treatment and expressed as percentage of the total neurons in the dish. In TG neurons, Sema3A induced higher percentage of cells with neurites, however quantification shows that this effect was not statistically significant. In DRG neurons the percentage of short, medium or long neurites was comparable between Sema3A and NGF treatment. (B) The average length and number of branches of long neurites was compared and significant differences observed when compared to untreated control neurons, but similar effects observed between Sema3A and NGF treatments. Values represent mean ± SD, experiments were performed in triplicate, each dish had an average of 200 neurons for DRG and 60 neurons for TG; all neurons in every dish were evaluated. * indicates p<0.05 vs Control.

Article Snippet: For immunofluorescence staining, section were fixed in ice cold methanol for 5 min, washed 3 times in PBS, blocked (2% BSA, 2.5% donkey serum in PBS) for 1h at room temperature and incubated overnight with 1:200 dilution of rabbit anti Sema3A antibody (cat # 21925, Proteintech, Rosemont, IL) at 4°C.

Techniques: Negative Control, Control

Journal: PLoS ONE

Article Title: Semaphorin3A induces nerve regeneration in the adult cornea-a switch from its repulsive role in development

doi: 10.1371/journal.pone.0191962

Figure Lengend Snippet: The neuronal promoting effects of Sema3A were tested on thy1-YFP mice subjected to superficial corneal epithelial debridement. The debridement of the epithelium also removes the sub basal nerve plexus without affecting the cornea nerves in the stroma. Insertion of an intrastromal pellet containing Sema3A or vehicle (see ) allows for the slow release into the cornea. (A ) Pellets containing vehicle (PBS) induced a discrete growth of sub basal nerves into the injured area. (B) However, addition of Sema3A induced faster regeneration of the superficial corneal nerves and higher nerve density was observed. (C) Quantification of nerve regeneration in the corneal injured area shows that Sema3A induced 3 folds higher nerve regeneration than control mice. White arrows = superficial nerves, yellow arrows = pellet. Values represent mean ± SEM, experiments were performed in triplicate, n = 5 per treatment, * indicates p<0.05 by ANOVA.

Article Snippet: For immunofluorescence staining, section were fixed in ice cold methanol for 5 min, washed 3 times in PBS, blocked (2% BSA, 2.5% donkey serum in PBS) for 1h at room temperature and incubated overnight with 1:200 dilution of rabbit anti Sema3A antibody (cat # 21925, Proteintech, Rosemont, IL) at 4°C.

Techniques: Control

Journal: Scientific Reports

Article Title: Matrilysin/MMP-7 Cleavage of Perlecan/HSPG2 Complexed with Semaphorin 3A Supports FAK-Mediated Stromal Invasion by Prostate Cancer Cells

doi: 10.1038/s41598-018-25435-3

Figure Lengend Snippet: Semaphorin 3A (Sema3A) antibody imitates Domain IV-3 activity on prostate cancer cells. Cell culture wells were spotted with control BSA and rabbit IgG (panel A), Domain IV-3 and rabbit IgG (panel B), BSA and Sema3A antibody (Ab) (panel C), or Domain IV-3 and Sema3A antibody (panel D) (inside the white dashed arc line). C4-2 cells were seeded and imaged 24 hours later. Scalebar is 250 µm. White asterisks indicate black fiduciary markers used to approximate the area of surface adsorbed substrate. ( B ) A western blot of C4-2 cells cultured for 24 hours on Domain IV-3 (lane 1), rabbit IgG (lane 2), Sema3A antibody (lane 3), or control BSA (lane 4). ( C ) Western blot of C4-2 cells cultured for 24 hours on control rabbit IgG or Sema3A antibody and probed for p-FAK, FAK, p-ERK, ERK1/2, and GAPDH. The Sema3A antibody as a substrate produces results akin to Domain IV-3 for C4-2 cells.

Article Snippet: Either control shRNA lentivirus (sc-108080) or SEMA3A directed shRNA lentivirus (sc-36470-V) (Santa Cruz Biotechnology, Santa Cruz, CA) was added to the well and incubated in standard conditions for 24 hours after which the viral media was removed and replaced with full media for an additional 24 hrs.

Techniques: Activity Assay, Cell Culture, Control, Western Blot

Journal: Scientific Reports

Article Title: Matrilysin/MMP-7 Cleavage of Perlecan/HSPG2 Complexed with Semaphorin 3A Supports FAK-Mediated Stromal Invasion by Prostate Cancer Cells

doi: 10.1038/s41598-018-25435-3

Figure Lengend Snippet: Sema3A binds Domain IV-3 and shRNA against Sema3A abrogates C4-2 cell clustering. ( A ) Domain IV-3 was adsorbed to well surfaces and subjected to a binding assay with Sema3A-Fc, Sema3A-Fc and Sema3A antibody, Sema3A antibody alone, and control IgG. Sema3A-Fc binds to Domain IV-3 and Sema3A antibody diminishes the binding signal significantly to 250 ng/mL (student’s unpaired t-test per concentration). ( B ) Blossom62 alignment of Ig 17 of perlecan (2 nd of Domain IV-3) and the Ig of Sema3A. C4-2 cells were seeded on Domain IV-3 after being transduced with either control shRNA (panel C) or Sema3A directed shRNA (panel D). ( E ) Dispersion index quantification between control and directed shRNA for each substrate type and amount. Significance values are student’s unpaired t-test between control and Sema3A shRNA. p-values: *<0.05, **<0.01, ***<0.001.

Article Snippet: Either control shRNA lentivirus (sc-108080) or SEMA3A directed shRNA lentivirus (sc-36470-V) (Santa Cruz Biotechnology, Santa Cruz, CA) was added to the well and incubated in standard conditions for 24 hours after which the viral media was removed and replaced with full media for an additional 24 hrs.

Techniques: shRNA, Binding Assay, Control, Concentration Assay, Transduction, Dispersion

Journal: Scientific Reports

Article Title: Matrilysin/MMP-7 Cleavage of Perlecan/HSPG2 Complexed with Semaphorin 3A Supports FAK-Mediated Stromal Invasion by Prostate Cancer Cells

doi: 10.1038/s41598-018-25435-3

Figure Lengend Snippet: Recombinant Sema3A-Fc is digested by MMP-7. ( A ) MMP-7 in silico digestion of Sema3A using SitePrediction online software. Shown is a schematic of Sema3A with its domains (sema, plexin/semaphorin/integrin (PSI), immunoglobulin (Ig)-like, basic). Each line represents a cleavage site with > 95% specificity. The bolded number is the rank in average score. Below rank average score is the amino acid sequence with the P1 cleavage site (cleaves at period). Amino acid sequence is based on UniProt ID Q14563, which includes the signal sequence not shown in the schematic. ( B ) Sema3A-Fc (0.75 µg) was incubated alone or with MMP-7 (0.08 µg) overnight. A silver stain and western blot against Sema3A indicate MMP-7 fragments Sema3A into multiple smaller sub bands including a 30 kDa band indicated by an arrow.

Article Snippet: Either control shRNA lentivirus (sc-108080) or SEMA3A directed shRNA lentivirus (sc-36470-V) (Santa Cruz Biotechnology, Santa Cruz, CA) was added to the well and incubated in standard conditions for 24 hours after which the viral media was removed and replaced with full media for an additional 24 hrs.

Techniques: Recombinant, In Silico, Software, Sequencing, Incubation, Silver Staining, Western Blot

Journal: Scientific Reports

Article Title: Matrilysin/MMP-7 Cleavage of Perlecan/HSPG2 Complexed with Semaphorin 3A Supports FAK-Mediated Stromal Invasion by Prostate Cancer Cells

doi: 10.1038/s41598-018-25435-3

Figure Lengend Snippet: Model of perlecan/Sema3A proposed interactions. In non-invasive prostate overgrowth or early cancer (PCa), intact perlecan is present in the immediate stroma surrounding Sema3A expressing prostatic cells. In this state (left), perlecan engages Sema3A, stabilizing its interaction with the neuropilin-1 and plexin complex. Plexin Ras-GAP activity favors a state of focal adhesion kinase (FAK) dephosphorylation and integrin deactivation. In this state, PCa cell-cell adhesion dominates cell-substratum adhesion, abrogating tissue invasion. As PCa progresses, active MMPs such as MMP-7 can cleave perlecan and/or Sema3A. Ras-GAP activity of plexins is lost, allowing FAK phosphorylation to occur along with subsequent downstream signaling (right). FAK signaling reactivates integrins to favor substrata-mediated dispersion, as seen to occur during tumor dyscohesion and metastasis. GAP = guanosine triphosphate (GTP)ase activation protein.

Article Snippet: Either control shRNA lentivirus (sc-108080) or SEMA3A directed shRNA lentivirus (sc-36470-V) (Santa Cruz Biotechnology, Santa Cruz, CA) was added to the well and incubated in standard conditions for 24 hours after which the viral media was removed and replaced with full media for an additional 24 hrs.

Techniques: Expressing, Activity Assay, De-Phosphorylation Assay, Phospho-proteomics, Dispersion, Activation Assay