Journal: Molecular Vision

Article Title: Semaphorin 3A blocks the formation of pathologic choroidal neovascularization induced by transforming growth factor beta

doi:

Figure Lengend Snippet: Effect of Sema3A on HUVEC proliferation. A , B , and C show the results and statistical analysis of HUVEC proliferation at different time points (24 h ( A ), 48 h ( B ), and 72 h ( C )). Sema3A inhibits HUVEC proliferation in general culture medium and under TGF-β-stimulated conditions. The experiment was performed in six wells and was repeated at least three times. The data are presented as the means±SEM. *p<0.05; **p<0.01.

Article Snippet: Sema3A (50,631-M01H; Sino Biologic, Inc., Beijing, China) was dissolved in PBS (Ca 2+ and Mg 2+ free) to 1 mg/ml as a stock solution and then diluted in Dulbecco's Modified Eagle Media (DMEM, Hyclone, Grand Island, NY) to the indicated concentrations.

Techniques:

Journal: Molecular Vision

Article Title: Semaphorin 3A blocks the formation of pathologic choroidal neovascularization induced by transforming growth factor beta

doi:

Figure Lengend Snippet: The effect of Sema3A on HUVEC migration. The cell nuclei, which were stained with DAPI, are shown as blue dots. The cells from five random fields were counted, and the average was used for the statistical analysis. A : Statistical analysis of the HUVEC migration study. Sema3A inhibits HUVEC migration in the general culture medium and in VEGF-stimulated conditions. B : 1% FBS-treated group. C : TGF-β-treated group. D : Sema3A (250 ng/ml)-treated group; E : Sema3A (500 ng/ml)-treated group. F : Sema3A (250 ng/ml)- and TGF-β-treated groups. G : Sema3A (500 ng/ml)- and TGF-β-treated groups. CTL represents the control group. *p<0.05; **p<0.01; ***p<0.0001.

Article Snippet: Sema3A (50,631-M01H; Sino Biologic, Inc., Beijing, China) was dissolved in PBS (Ca 2+ and Mg 2+ free) to 1 mg/ml as a stock solution and then diluted in Dulbecco's Modified Eagle Media (DMEM, Hyclone, Grand Island, NY) to the indicated concentrations.

Techniques: Migration, Staining

Journal: Molecular Vision

Article Title: Semaphorin 3A blocks the formation of pathologic choroidal neovascularization induced by transforming growth factor beta

doi:

Figure Lengend Snippet: Effect of Sema3A on HUVEC apoptosis. Sema3A induced HUVEC apoptosis in the general culture medium and in the TGF-β-stimulated conditions. FITC-Annexin V staining was measured at the treatment time points of 24, 48, and 72 h. A : Statistical analysis of the HUVEC apoptosis study. B : Representative image of the 1% FBS-treated group. C : Representative image of the TGF-β-treated group. D : Representative image of the Sema3A (250 ng/ml)-treated group. E : Representative image of the Sema3A (500 ng/ml)-treated group. F : Representative image of the Sema3A (250 ng/ml)- and TGF-β-treated groups. G : Representative image of the Sema3A (500 ng/ml)- and TGF-β-treated groups. UR=late apoptotic cells; LR=early apoptotic cells; UR+LR=total apoptotic cells. The experiment was repeated at least three times. The data are presented as the means±SEM. *p<0.05; **p<0.01.

Article Snippet: Sema3A (50,631-M01H; Sino Biologic, Inc., Beijing, China) was dissolved in PBS (Ca 2+ and Mg 2+ free) to 1 mg/ml as a stock solution and then diluted in Dulbecco's Modified Eagle Media (DMEM, Hyclone, Grand Island, NY) to the indicated concentrations.

Techniques: Staining

Journal: Molecular Vision

Article Title: Semaphorin 3A blocks the formation of pathologic choroidal neovascularization induced by transforming growth factor beta

doi:

Figure Lengend Snippet: Effect of Sema3A on HUVEC tube formation. The Matrigel assay was used to evaluate the angiogenic effect in Sema3A-treated HUVECs. This assay measures the ability of endothelial cells to form capillary-like structures. A : Statistical analysis of the HUVEC tube formation study. B : 1% fetal bovine serum (FBS)-treated group. C : TGF-β culture group. D : Sema3A (250 ng/ml)-treated group. E : Sema3A (500 ng/ml)-treated group. F : Sema3A (250 ng/ml)- and TGF-β-treated groups. G : Sema3A (500 ng/ml)- and TGF-β-treated groups. All of the pictures (panel B to G ) were taken at 10× magnification under a Zeiss light microscope. All of the data were measured at least three times and are presented as the means±SEM. *p<0.05; **p<0.01.

Article Snippet: Sema3A (50,631-M01H; Sino Biologic, Inc., Beijing, China) was dissolved in PBS (Ca 2+ and Mg 2+ free) to 1 mg/ml as a stock solution and then diluted in Dulbecco's Modified Eagle Media (DMEM, Hyclone, Grand Island, NY) to the indicated concentrations.

Techniques: Matrigel Assay, Light Microscopy

Journal: Molecular Vision

Article Title: Semaphorin 3A blocks the formation of pathologic choroidal neovascularization induced by transforming growth factor beta

doi:

Figure Lengend Snippet: The TGF-β concentration in Sema3A-treated HUVECs. The concentration of free TGF-β1 in the supernatant was measured using an ELISA kit. The statistical analysis of the free TGF-β measured with ELISA at 48 h is presented. Sema3A inhibited the response of TGF-β. Each experiment was performed in six wells and was repeated at least three times. The data are presented as the means±SEM. **p<0.01; ***p<0.0001.

Article Snippet: Sema3A (50,631-M01H; Sino Biologic, Inc., Beijing, China) was dissolved in PBS (Ca 2+ and Mg 2+ free) to 1 mg/ml as a stock solution and then diluted in Dulbecco's Modified Eagle Media (DMEM, Hyclone, Grand Island, NY) to the indicated concentrations.

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Molecular Vision

Article Title: Semaphorin 3A blocks the formation of pathologic choroidal neovascularization induced by transforming growth factor beta

doi:

Figure Lengend Snippet: CNV leakage with FA assessment of the CNV mouse retina. Angiographic analysis of CNV leakage 14 days after laser photocoagulation in the different treatment groups and in the control group. A : The normal control. B : The CNV control. C : The Sema3A (10 ng/ml)-treated group. D : The Sema3A (100 ng/ml)-treated group. E : The results and the statistical analysis of the CNV leakage by FA. The data are presented as the means±SEM. *p<0.05; **p<0.01.

Article Snippet: Sema3A (50,631-M01H; Sino Biologic, Inc., Beijing, China) was dissolved in PBS (Ca 2+ and Mg 2+ free) to 1 mg/ml as a stock solution and then diluted in Dulbecco's Modified Eagle Media (DMEM, Hyclone, Grand Island, NY) to the indicated concentrations.

Techniques:

Journal: Molecular Vision

Article Title: Semaphorin 3A blocks the formation of pathologic choroidal neovascularization induced by transforming growth factor beta

doi:

Figure Lengend Snippet: The VEGF and TGF-β concentrations in Sema3A-treated retina-choroid complexes. The VEGF and TGF-β1 concentrations in the retina-choroid complexes were measured using an ELISA kit. The statistical analysis of the free VEGF and TGF-β measured at 14 days after Sema3A treatment is presented. Each experiment was repeated at least three times. The data are presented as the means±SEM. *p<0.05; **p<0.01.

Article Snippet: Sema3A (50,631-M01H; Sino Biologic, Inc., Beijing, China) was dissolved in PBS (Ca 2+ and Mg 2+ free) to 1 mg/ml as a stock solution and then diluted in Dulbecco's Modified Eagle Media (DMEM, Hyclone, Grand Island, NY) to the indicated concentrations.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Molecular Vision

Article Title: Semaphorin 3A blocks the formation of pathologic choroidal neovascularization induced by transforming growth factor beta

doi:

Figure Lengend Snippet: Effect of Sema3A on SMAD2/3 and phosphorylated SMAD2/3 in HUVECs and in the CNV mouse model. Immunoblot images ( A , D ) and statistical analyses ( B , C , E , and F ) for the SMAD2/3 and p-SMAD2/3 signaling pathways. The results show that Sema3A inhibits SMAD2/3 and p-SMAD2/3 in HUVECs and in the CNV mouse model. The western blot analyses were repeated three times, and qualitatively similar results were obtained. The data are presented as the means±SEM. *p<0.05; **p<0.01.

Article Snippet: Sema3A (50,631-M01H; Sino Biologic, Inc., Beijing, China) was dissolved in PBS (Ca 2+ and Mg 2+ free) to 1 mg/ml as a stock solution and then diluted in Dulbecco's Modified Eagle Media (DMEM, Hyclone, Grand Island, NY) to the indicated concentrations.

Techniques: Western Blot

Journal: Molecular Vision

Article Title: Semaphorin 3A blocks the formation of pathologic choroidal neovascularization induced by transforming growth factor beta

doi:

Figure Lengend Snippet: Effect of Sema3A on phosphorylated p38 MAPK, ERK1/2, and JNK in the CNV mouse model. Immunoblot ( A ) and statistical ( B ) analyses of the p-p38 MAPK, p-ERK1/2, and p-JNK signaling pathways. The results show that Sema3A inhibits p-p38 MAPK, p-ERK1/2, and p-JNK in the CNV mouse model. The western blot analyses were repeated three times, and qualitatively similar results were obtained. The data are presented as the means±SEM. *p<0.05; **p<0.01.

Article Snippet: Sema3A (50,631-M01H; Sino Biologic, Inc., Beijing, China) was dissolved in PBS (Ca 2+ and Mg 2+ free) to 1 mg/ml as a stock solution and then diluted in Dulbecco's Modified Eagle Media (DMEM, Hyclone, Grand Island, NY) to the indicated concentrations.

Techniques: Western Blot

Journal: Nature Communications

Article Title: Neuropilin 1 regulates bone marrow vascular regeneration and hematopoietic reconstitution

doi: 10.1038/s41467-021-27263-y

Figure Lengend Snippet: a Heatmap of semaphorin (Sema) gene expression in irradiated and non-irradiated (non-IRR) BM ECs isolated before and after irradiation ( n = 3/condition). b qRT-PCR analysis of Sema and Nrp expression in VE-cad + BM ECs from Non-IRR and 500 cGy-irradiated mice ( n = 6/condition from n = 10,000 cells/replicate; **** p < 0.0001). Data normalized to non-IRR sample. Horizontal bars represent means. c qRT-PCR of Sema3a expression in BM hematopoietic cell populations, LepR + stromal cells and CD31 + Sca-1 − sinusoidal BM ECs (sBMECs) and CD31 + Sca-1 + arterial BM ECs (aBMECs; n = 4–12 replicates/condition; **** p < 0.0001). Data normalized to KSL cells. d qRT-PCR of Sema3a expression in aBMECs and sBMECs before and +24 h following 500 cGy ( n = 6/group; aBMEC: *** p = 0.0008, sBMEC: n.s. p = 0.72). Data normalized to non-IRR. e qRT-PCR of Nrp1 expression in BM hematopoietic cell populations, LepR + stromal cells and ECs ( n = 4–12 replicates/group; **** p < 0.0001). Data normalized to KSL cells. f SEMA3A concentration in the BM of mice following 500 cGy quantified by ELISA ( n = 8 mice; 6 h, d1, d2: **** p < 0.0001, d7: *** p = 0.0001, d14: *** p = 0.0007). Dotted line represents BM SEMA3A concentration in non-IRR mice. g Confocal images of femurs from C57BL/6J mice before and +72 h following 500 cGy. Expression of SEMA3A (yellow) in VE-Cad + BM ECs (magenta) and CD31 + BM ECS (green) is shown (white arrows). Nuclei labeled with DAPI (blue). Representative images from n = 3 experiments, Scale bar 50 µm; magnified scale bar, 20 µm; max z -projections shown. h Expression of NRP1 (yellow) in VE-Cad + BM ECs (magenta) and CD31 + BM ECS (green) is shown (white arrows) in same conditions. Representative images from n = 3 experiments. i Representative gating, histogram, and j quantification of NRP1 expression on Non-IRR and 500 cGy-irradiated BM ECs ( n = 5/group, **** p < 0.0001, aBMEC: p = 0.8256). k NRP1 MFI within BM EC sub-populations before and +24 h post-500 cGy ( n = 5/group, **** p < 0.0001). Data assessed by Holm-Sidak’s multiple comparison two-sided t -test after two-way ANOVA ( b , d , f , j , k ) and the Holm-Sidak’s multiple comparison two-sided t -test after one-way ANOVA ( c , e ). Data presented as mean values +/− SEM. ** p < 0.01, *** p < 0.001, **** p < 0.0001. Source data are provided as a Source Data file. .

Article Snippet: The next day, plates were washed with 1× wash buffer (ab128573, Abcam, Waltham, MA) and blocked with 200 μl/well of 1% bovine serum albumin (BSA) in PBS containing 0.05% NaN 3 at room temperature (RT) for 3 h. Subsequently, wells were washed three times with 1× wash buffer (ab128573, Abcam, Waltham, MA) and mouse SEMA3A Fc Chimera Protein, CF (5926-S3 R&D systems, Minneapolis, MN) or human SEMA3A Fc Chimera Protein (R&D, 1250-S3) was added to the NRP1-coated wells, at increasing concentrations of 0, 1, 2, 4, 8, 16, 32, 128 ng/ml and incubated for 2 h at RT.

Techniques: Gene Expression, Irradiation, Isolation, Quantitative RT-PCR, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Labeling, Comparison

Journal: Nature Communications

Article Title: Neuropilin 1 regulates bone marrow vascular regeneration and hematopoietic reconstitution

doi: 10.1038/s41467-021-27263-y

Figure Lengend Snippet: a qRT-PCR analysis of Sema3a and Nrp1 expression in BM ECs from mice at +24 h following 5-FU chemotherapy. For each gene, expression is normalized to vehicle treatment ( n = 5/condition; Sema3a : p < 0.0001, Nrp1 : p = 0.1291). b Representative images of VE-cadherin + (magenta) , CD31 + (green) BM vessels in femur sections from untreated control mice and at day +7 following 5-FU chemotherapy and treatment with anti-NRP1 or IgG. Nuclei are stained with DAPI (blue). Scale bar, 50 µm; magnified view scale bar, 20 µm. c BM cell counts at day +7 following 5-FU chemotherapy and treatment with anti-NRP1 or IgG ( n = 5 mice/group, p = 0.0219). d At left, representative flow cytometric analysis of CD45 − VE-cad + BM ECs within BM lin − cells from control mice and at day +7 following 5-FU chemotherapy and anti-NRP1 or IgG treatment. At right, percentages and numbers of BM ECs at day +7 following 5-FU chemotherapy and the treatments shown. Dotted lines represent numbers in control mice ( n = 5 mice/group; % BM ECs p < .0001; number BM ECs p = 0.0008). e At left, representative histograms show % activated caspase 3/7 + cells within lin − CD45 − VE-cad + BM ECs at day +7 following 5-FU. At right, % activated caspase 3/7 + BM ECs at day +7 ( n = 5/group; p < .0001). Dotted line represents percentages of activated caspase 3/7 + cells within lin − CD45 − VE-cad + BM ECs in non-IRR controls. f PB WBC, NEU, and LYMPHs at day +7 following 5-FU and the treatments shown ( n = 5 mice/group; p < 0.0001). g At left, flow cytometry of BM KSL cells at day +7 following 5-FU and the treatments shown. At right, % BM KSL cells ( n = 5 mice/group; p = 0.011). h Colony forming unit quantification from 30,000 BM cells isolated from mice at day +7 following 5-FU and IgG or anti-NRP1 treatment ( n = 4 replicates, mean values +/− SEM; GM: p < 0.0001; GEMM: p = 0.0201). Statistics show GM and GEMM comparisons. a , f , h Two-way ANOVA followed by Holm-Sidak’s multiple comparison two-sided t -tests; c , d , g Student’s unpaired two-sided t -test. * p < 0.05, *** p < 0.001, **** p < 0.0001. Source data provided as a Source Data file.

Article Snippet: The next day, plates were washed with 1× wash buffer (ab128573, Abcam, Waltham, MA) and blocked with 200 μl/well of 1% bovine serum albumin (BSA) in PBS containing 0.05% NaN 3 at room temperature (RT) for 3 h. Subsequently, wells were washed three times with 1× wash buffer (ab128573, Abcam, Waltham, MA) and mouse SEMA3A Fc Chimera Protein, CF (5926-S3 R&D systems, Minneapolis, MN) or human SEMA3A Fc Chimera Protein (R&D, 1250-S3) was added to the NRP1-coated wells, at increasing concentrations of 0, 1, 2, 4, 8, 16, 32, 128 ng/ml and incubated for 2 h at RT.

Techniques: Quantitative RT-PCR, Expressing, Gene Expression, Control, Staining, Flow Cytometry, Isolation, Comparison

Journal: Nature Communications

Article Title: Neuropilin 1 regulates bone marrow vascular regeneration and hematopoietic reconstitution

doi: 10.1038/s41467-021-27263-y

Figure Lengend Snippet: a (Left) flow cytometry of activated caspase 3/7 + BM ECs in wild-type (WT) and Nrp1 VEGF− mice at day +5 following 500 cGy and the treatments shown; (Right) % activated caspase 3/7 + BM ECs ( n = 5/group; **** p < 0.0001; *** p = 0.0003). b (Left) Donor CD45.2 + cell engraftment at 20 weeks in the PB of CD45.1 + mice competitively transplanted with BM cells from 500 cGy—irradiated Nrp1 VEGF− mice treated with anti-NRP1 or IgG; (Right) % donor CD45.2 + , Mac1 + /Gr1 + , B220 + , and CD3 + cells at 20 weeks ( n = 5–6/group; total: p = 0.0346; myeloid: p = 0.0325; T cells: p = 0.0195). c (Left) Histograms of p-Cdk5 + BM ECs at 1 h following 800 cGy and the treatments shown; (Right) %p-p53 + BM ECs. Horizontal lines show gating for p-Cdk5 + cells ( n = 5 replicates, Non-IRR; n = 6 replicates for IRR, IRR + SEMA3A, IRR + SEMA3A + anti-NRP1; non-IRR vs. IRR: p < 0.0001; IRR vs. IRR + SEMA3A: p = 0.0005; IRR vs. IRR + SEMA3A + Anti-NRP1 p < 0.0001). d % activated caspase 3/7 + BM ECs at 24 h following 800 cGy and treatment with SEMA3A, SEMA3A + anti-NRP1 or SEMA3A + roscovitine (ROSC), 10 ng/ml ( n = 4–8 replicates; non-IRR vs. IRR: p < 0.0001; IRR vs. IRR + SEMA3A: p = 0.0007; IRR vs. IRR + SEMA3A + Anti-NRP1 p < 0.0001; IRR vs. IRR + SEMA3A + Anti-NRP1 + ROSC p < 0.0001). e At left, histograms of p-p53 + BM ECs at 1 h following 800 cGy and treatment with SEMA3A, SEMA3A + anti-NRP1, or SEMA3A + ROSC. At right, % p-p53 + BM ECs are shown. Black horizontal lines show gating for p-p53 + cells ( n = 6 replicates, Non-IRR; n = 9 replicates for IRR and IRR + SEMA3A; n = 5 replicates for IRR + SEMA3A + anti-NRP1; n = 4 replicates for IRR + SEMA3A + ROSC; p < 0.0001 for all comparisons). f At left, flow cytometry for activated caspase 3/7 + BM ECs from p53 −/− and p53 + /+ mice at 24 h following irradiation with 800 cGy and the treatments shown. At right, % activated caspase 3/7 + BM ECs are shown ( n = 5 replicates/group, p < 0.0001). g Puma expression in BM ECs at 6 h following 800 cGy and the treatments shown ( n = 4/condition; **** p < 0.0001, * p = 0.0142, ** p = 0.0025, *** p = 0.0009). a , c , d , e One-way ANOVA followed by Holm-Sidak’s two-sided unpaired t -tests, f Two-way ANOVA followed by Holm-Sidak’s two-sided unpaired t -test, g Brown–Forsythe ANOVA followed by two-sided unpaired t -tests with Welch’s correction, b Two-sided Mann–Whitney test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Source Data file provided.

Article Snippet: The next day, plates were washed with 1× wash buffer (ab128573, Abcam, Waltham, MA) and blocked with 200 μl/well of 1% bovine serum albumin (BSA) in PBS containing 0.05% NaN 3 at room temperature (RT) for 3 h. Subsequently, wells were washed three times with 1× wash buffer (ab128573, Abcam, Waltham, MA) and mouse SEMA3A Fc Chimera Protein, CF (5926-S3 R&D systems, Minneapolis, MN) or human SEMA3A Fc Chimera Protein (R&D, 1250-S3) was added to the NRP1-coated wells, at increasing concentrations of 0, 1, 2, 4, 8, 16, 32, 128 ng/ml and incubated for 2 h at RT.

Techniques: Flow Cytometry, Irradiation, Expressing, MANN-WHITNEY

Journal: Oncogene

Article Title: Extracellular vesicle-transported Semaphorin3A promotes vascular permeability in glioblastoma.

doi: 10.1038/onc.2015.317

Figure Lengend Snippet: Figure 2. EV-transported Sema3A enhances vascular permeability through NRP1. (a) Sema3A concentrations were measured by ELISA in purified EVs from HEK293T, GSC#1, #4 and #9. (b) Flow-cytometry analysis of Sema3A surface expression in purified EVs from GSC#1, #4 and #9. PE-conjugated isotype control (IgG) is shown in grey. (c) Confocal analysis of purified GSC#1-shed EVs stained with Alix (green) and Sema3A (S3A, green). Nucleic acid staining is shown in blue (DAPI). Scale bar: 500 nm. (d) Electronic microscopy analysis of purified GSC#4-secreted EVs stained by immunogold for CD63 and Sema3A. Scale bars: 50 nm. (e) Western-blot analysis for Sema3A, CD9 and Rab4 in purified GSC#4-secreted EVs (EV#4) either untreated (−) or subjected to proteinase K treatment (Prot K, +). (f) EVs were purified from conditioned medium of GSC#4 that received either non-silencing RNA (siC, blue bar) or siRNA targeting Sema3A (siS3A, black bar). Sema3A concentrations were measured by ELISA. **Po0.01. (g, h) EVs as prepared in (f) were administered to measure endothelial permeability g) in vitro and (h) in vivo (n = 6). Results are expressed as fold change to untreated conditions ( −and PBS, respectively). Insert panel shows RT-PCR for Sema3A and Actin (ACTB) in GSC#4. *Po0.05; **Po0.01. (i) Permeability assays were performed in non-silencing (siC) or siRNA targeting NRP1 (siNRP1)-transfected endothelial cells (ECs). Two days after transfection, ECs were treated with purified GSC#4-secreted EVs (EV#4, green) or control vehicle (Ctrl, red). Insert panel shows RT-PCR for NRP1 and Actin (ACTB), to assess siNRP1 efficiency in ECs. **Po0.01. (j) ECs were pre-treated with blocking antibodies (25 mg/ml) preventing Sema3A (aNRP1A) or VEGF-A (aNRP1B) binding to NRP1 receptor. Human immunoglobulins (IgG) were used as a control with the same concentration as blocking antibodies. ECs were then exposed to purified GSC#4-shed EVs. **Po0.01. (k) Mice received intradermic injections of blocking (175 mg/ml) and control antibodies (IgG) together with purified GSC#4-secretd EVs. Vascular permeability was measured using in vivo Miles assay (n = 5). **Po0.01; ***Po0.001. Each panel is representative of at least three independent experiments.

Article Snippet: Sema3A protein concentration was then estimated by ELISA as per the manufacturer’s instruction (Cusabio, Clinisciences, Nanterre, France).

Techniques: Permeability, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Expressing, Control, Staining, Microscopy, Western Blot, In Vitro, In Vivo, Reverse Transcription Polymerase Chain Reaction, Transfection, Blocking Assay, Binding Assay, Concentration Assay

Journal: Oncogene

Article Title: Extracellular vesicle-transported Semaphorin3A promotes vascular permeability in glioblastoma.

doi: 10.1038/onc.2015.317

Figure Lengend Snippet: Figure 3. Tumour-derived EV-transported Sema3A enhances vascular permeability. (a–c) EVs were purified from conditioned medium of GSC#4 that received either non-silencing RNA (siC) or Sema3A-targeting (siS3A), and applied to the left hemisphere of mouse brain. Vehicle (PBS) served as a control. (a) Evans blue extravasation is shown in OCT-included brains. Arrow indicated the site of EV dropping (left hemisphere). (b) Intensity of Evans blue extravasation was quantified in left and right hemispheres, including all brain areas (whole), only cerebrum (crbum) or cerebellum (crbellum). *Po0.05; **Po0.01; ***Po0.001. (c) Confocal analysis of the red blood cell marker Ter119 (red), Claudin5 (Cldn5, green) and Evans Blue (grey) in frozen brain sections. Nucleic acid staining is shown in blue (DAPI). Scale bar: 40 μm. Graph shows the quantification of Ter119 signal intensity from confocal analysis. **Po0.01. (d) Frozen tissue sections from subcutaneous ectopic and intracranial orthotopic GSC#9 xenografts were stained with Pecam (green) and Ter119 (red). Nucleic acid staining is shown in blue (DAPI). Scale bar: 20 μm. (e) Circulating blood EVs were purified from non-injected (non-tum EV, blue) and GSC-xenografted (xeno EV, red) mice and were analysed by flow cytometry for mouse (m) and human (h) CD63. Respective isotype controls (IgG) are shown in grey. Percentage represents the number of positive events for hCD63. (f) Human Sema3A (hSema3A) concentrations were measured by ELISA in circulating EVs purified from sera of non-injected ( −, light orange) and ectopically (ecto.) or orthotopically (ortho.) GSC xenografted (GSC, orange) mice. *Po0.05; **Po0.01. Each panel is representative of at least three independent experiments.

Article Snippet: Sema3A protein concentration was then estimated by ELISA as per the manufacturer’s instruction (Cusabio, Clinisciences, Nanterre, France).

Techniques: Derivative Assay, Permeability, Control, Marker, Staining, Injection, Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Oncogene

Article Title: Extracellular vesicle-transported Semaphorin3A promotes vascular permeability in glioblastoma.

doi: 10.1038/onc.2015.317

Figure Lengend Snippet: Figure 4. Peripheral blood-isolated EVs from glioblastoma patients promote vascular permeability through a Sema3A/NRP1-dependent mechanism. Circulating EVs were purified from healthy donor and GBM patient sera. (a) Representative FSC/SSC dot-plot of the EV population, resuspended in filtered PBS, and analysed by flow cytometry together with 10-μm-calibrated beads (cal). EVs were further analysed by flow cytometry for Annexin V (AnnV, blue) and CD63 (blue) relative to their respective controls (Ctl, red). (b) Evans blue dye extravasation (Miles assay) of intradermic injected mice with purified EVs from healthy donors (Healthy, green) or GBM patients (GBM, orange). *Po0.05. (c) Intensity of Evans blue extravasation was quantified in left and right hemispheres, including all brain areas (whole), only cerebrum (crbum) or cerebellum (crbellum). **Po0.01; ***Po0.001. (d) Sema3A concentrations were measured by ELISA in purified-EV from healthy donors (green, n = 15) or GBM patient sera (red, n = 4). ***Po0.001. (e) Mice received intradermic injections of blocking (aNRP1A and aNRP1B) and control antibodies (IgG) together with purified GBM patient sera-derived EVs (GBM-EVs). Vascular permeability was measured using in vivo Miles assay. **Po0.01; ***Po0.001. (f) Graphs show the quantification of Evans blue signal intensity from confocal analysis of mouse epidermis after Miles assay. *Po0.05; **Po0.01. Each panel is representative of at least three independent experiments.

Article Snippet: Sema3A protein concentration was then estimated by ELISA as per the manufacturer’s instruction (Cusabio, Clinisciences, Nanterre, France).

Techniques: Isolation, Permeability, Cytometry, Injection, Enzyme-linked Immunosorbent Assay, Blocking Assay, Control, Derivative Assay, In Vivo

Journal: Development (Cambridge, England)

Article Title: Semaphorin signaling facilitates cleft formation in the developing salivary gland.

doi: 10.1242/dev.005066

Figure Lengend Snippet: Fig. 3. Sema3A and Sema3C additively promote SMG cleft formation. (A) Sema3A and Sema3C are the only class 3 semaphorins that could promote cleft formation in the SMG co-culture assays. Representative photographs of the growth of SMG explants co-cultured with class 3 semaphorins were shown. The bar graph summarizes the ratios of the number of terminal buds in each co-culture to the number of the terminal buds in the control culture (n7). The number of terminal buds at the presence of Sema3A or Sema3C was almost doubled. (B) Treatment of antisense ODNs against Sema3A or Sema3C specifically reduced the number of terminal buds in the SMG cultured ex vivo. Representative explants are shown. The bar graph summarizes five independent experiments. The terminal bud number was significantly further reduced when both Sema3A antisense ODNs and Sema3C antisense ODNs were added together into the culture. Paired t-test: *, P<0.05; **, P<0.01. (C) Sema3A and Sema3C additively promoted bud formation in a concentration-dependent manner. In the SMG co-culture experiments, Sema3-transfected COS cells were serially diluted to test the synergistic effects. Fold dilution in the co-culture is indicated as the Sema3-transfected COS cells diluted with mock-transfected COS cells. (a) Additive effects of Sema3A and Sema3C were most obvious at the lower concentrations of semaphorins. The effects were saturated at 1:1 dilution. (b) The amounts of semaphorin proteins present in each condition were assayed by western blotting. Gradual decreases of Sema3A or Sema3C in the serial dilutions were observed. Tubulin: internal loading control. (c) The bar graph summarizes four independent experiments. Scale bars: 100 m. C, scrambled sequence.

Article Snippet: The human recombinant SEMA3A peptide (R&D systems) was added at concentrations of 10, 25, 50, 100 or 150 ng/ml, respectively.

Techniques: Co-Culture Assay, Cell Culture, Control, Ex Vivo, Concentration Assay, Transfection, Western Blot, Sequencing

Journal: Development (Cambridge, England)

Article Title: Semaphorin signaling facilitates cleft formation in the developing salivary gland.

doi: 10.1242/dev.005066

Figure Lengend Snippet: Fig. 4. Functional Npn1 is required for the Sema3A- and Sema3C-mediated SMG branching morphogenesis in mice. (A) Anti-Npn1 antibody dose-dependently abolished SMG cleft formation promoted by Sema3A or Sema3C in the SMG co-cultures. Complete inhibition could be achieved in the presence of 5 g/ml neutralizing antibody. Representative explants are shown and the summary of six independent experiments is shown in the bar graph. Paired t-test: *, P<0.05; **, P<0.01. (B) Npn1-AP fusion proteins, but not AP proteins, blocked Sema3A-mediated branching activity in the SMG co-cultures. Note that Npn1-AP fusion protein alone could block the endogenous branching activities (Mock) (upper panels). By contrast, application of Npn2-Fc fusion proteins in the co-cultures had no effects on the SMG branching activity (lower panels). Representative explants were shown and the summary of five independent experiments is shown in the bar graph. Paired t-test: *, P<0.05; **, P<0.01. (C) Sema3A-AP bound the epithelial buds in the SMG cultured ex vivo for 24 hours. As a control, when the Sema3A-AP conditioned medium was depleted of the AP-fusion proteins by pre-incubation with Npn1-transfected COS cells, the binding activity on the epithelial buds was greatly reduced. (D) Sema3A mRNA was detected in the SMG epithelial buds cultured ex vivo (a) and in E15.5 embryonic SMG (b). The expression was distributed as a gradient with the highest level at the front end of the terminal bud. Immunofluorescence staining of E-cadherin highlighted the epithelial buds in a. Area within dashed line, epithelial bud. (E) Semi-quantitative RT-PCR analysis confirmed that Sema3A transcript was mainly in the SMG epithelium. Scale bars: 100 m. C, mock-transfected COS cells; Epi, epithelium; IF, immunofluorescence; ISH, RNA in situ hybridization; M, mesenchyme.

Article Snippet: The human recombinant SEMA3A peptide (R&D systems) was added at concentrations of 10, 25, 50, 100 or 150 ng/ml, respectively.

Techniques: Functional Assay, Inhibition, Activity Assay, Blocking Assay, Cell Culture, Ex Vivo, Control, Incubation, Transfection, Binding Assay, Expressing, Immunofluorescence, Staining, Quantitative RT-PCR, RNA In Situ Hybridization

Journal: Development (Cambridge, England)

Article Title: Semaphorin signaling facilitates cleft formation in the developing salivary gland.

doi: 10.1242/dev.005066

Figure Lengend Snippet: Fig. 5. VEGF is not required for Npn1-mediated cleft formation in mice. (A) Whole-mount immunohistochemistry showed that VEGF and its receptors, Flt1 and Flk1, did not express in the epithelial buds of the SMG. By contrast, Fgfr2 was expressed in the SMG epithelial buds as previously reported. (B) Neither exogenous VEGF nor VEGF antibody (VEGF Ab) affected normal branching morphogenesis of the SMG cultured ex vivo. The bar graph summarizes five independent experiments. (C) VEGF had no effects on the branching activities promoted by Sema3A. The bar graph summarizes four independent experiments. (D) VEGF could not change effects caused by application of Npn1-neutralizing antibody at sub-optimal concentration (2 g/ml) in the SMG ex vivo cultures. The bar graph summarizes five independent experiments. Scale bars: 100 m.

Article Snippet: The human recombinant SEMA3A peptide (R&D systems) was added at concentrations of 10, 25, 50, 100 or 150 ng/ml, respectively.

Techniques: Immunohistochemistry, Cell Culture, Ex Vivo, Concentration Assay

Journal: Development (Cambridge, England)

Article Title: Semaphorin signaling facilitates cleft formation in the developing salivary gland.

doi: 10.1242/dev.005066

Figure Lengend Snippet: Fig. 6. Plexin A2 and plexin D1 are required for class 3 semaphorin- mediated cleft formation. (A) SMG cleft formation was reduced in the SMG ex vivo cultures treated with antisense ODNs against plexin A2 or plexin D1 for 48 hours. No effects were observed in the SMG explants dissected from knockout mouse mutants of plexin A3 or plexin A4, or in the SMG explants treated with antisense ODNs against other plexins. Representative explants are shown in a and the summary of five independent experiments is shown as a bar graph in b. Paired t-test: *, P<0.05 while compared with the controls. (B) SMG cleft formation promoted by Sema3A in the SMG co- cultures was partially abolished by the treatment of antisense ODNs against plexin A2 or plexin D1, but not by sense ODNs. Representative photographs were taken from 24-hour co-cultures (a). A summary of seven independent experiments is shown in b. Scale bars: 100 m. AS, antisense ODNs; KO, knockout mutants; S, sense ODNs.

Article Snippet: The human recombinant SEMA3A peptide (R&D systems) was added at concentrations of 10, 25, 50, 100 or 150 ng/ml, respectively.

Techniques: Ex Vivo, Knock-Out

Journal: Development (Cambridge, England)

Article Title: Semaphorin signaling facilitates cleft formation in the developing salivary gland.

doi: 10.1242/dev.005066

Figure Lengend Snippet: Fig. 8. Sema3A restricts migration of cultured SMG epithelial cells. (A) The migration of mouse epithelial cells in mesenchyme-free SMG explant culture was restricted by synthetic Sema3A peptide (100 ng/ml). (B) The extent of epithelial cell migration is summarized at three time points, as indicated. The migration ratios were obtained from dividing the area measured at each time point by the area measured at 0 hours. (C) Sema3A caused cytoplasmic constriction of the SMG epithelial cells. Dissociated epithelial cells from E13.5 SMG were cultured for 2 days and then treated with synthetic Sema3A or scrambled peptide for another 1 or 2 days. Cells were fixed with paraformaldehyde and stained with rhodamine phalloidin. Scale bar: 100 m.

Article Snippet: The human recombinant SEMA3A peptide (R&D systems) was added at concentrations of 10, 25, 50, 100 or 150 ng/ml, respectively.

Techniques: Migration, Cell Culture, Staining

Journal: PLoS ONE

Article Title: Extract of Scutellaria baicalensis induces semaphorin 3A production in human epidermal keratinocytes

doi: 10.1371/journal.pone.0250663

Figure Lengend Snippet: A list of herbal plants used in screening of Sema3A inducers.

Article Snippet: Cells cultured for 6 or 24 hours were lysed in M-PER mammalian protein extraction reagent and protease inhibitor cocktail (Thermo Scientific, Waltham MA, USA), then Sema3A levels in the cell lysate were measured using a Human Sema3A ELISA kit (Elabscience, Houston, TX, USA).

Techniques:

Journal: PLoS ONE

Article Title: Extract of Scutellaria baicalensis induces semaphorin 3A production in human epidermal keratinocytes

doi: 10.1371/journal.pone.0250663

Figure Lengend Snippet: A summary of Sema3A reporter gene activity by herbal plant extracts.

Article Snippet: Cells cultured for 6 or 24 hours were lysed in M-PER mammalian protein extraction reagent and protease inhibitor cocktail (Thermo Scientific, Waltham MA, USA), then Sema3A levels in the cell lysate were measured using a Human Sema3A ELISA kit (Elabscience, Houston, TX, USA).

Techniques: Activity Assay

Journal: PLoS ONE

Article Title: Extract of Scutellaria baicalensis induces semaphorin 3A production in human epidermal keratinocytes

doi: 10.1371/journal.pone.0250663

Figure Lengend Snippet: NHEKs were transfected with pGL3- Sema3A . After transfection, the cells were incubated with five plant extracts, namely No. 2, 7, 10, 13, and 76 at a final concentration of 5, 15, and 50 μg/mL for 48 hours at 37°C. Cell lysates were subjected to luciferase activity measurement as described in Materials and methods. All results are expressed as mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (one-way ANOVA followed by Dunnett’s test).

Article Snippet: Cells cultured for 6 or 24 hours were lysed in M-PER mammalian protein extraction reagent and protease inhibitor cocktail (Thermo Scientific, Waltham MA, USA), then Sema3A levels in the cell lysate were measured using a Human Sema3A ELISA kit (Elabscience, Houston, TX, USA).

Techniques: Transfection, Incubation, Concentration Assay, Luciferase, Activity Assay

Journal: PLoS ONE

Article Title: Extract of Scutellaria baicalensis induces semaphorin 3A production in human epidermal keratinocytes

doi: 10.1371/journal.pone.0250663

Figure Lengend Snippet: NHEKs were incubated with five plant extracts, samples No. 2, 7, 10, 13, and 76 at a final concentration of 50 μg/mL for 3 and 6 hours at 37°C. Expression of Sema3A mRNA was examined using quantitative real-time PCR analysis. The levels of Sema3A mRNA were normalized relative to those of RPS18 . All results are expressed as mean ± S.D. of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (one-way ANOVA followed by Dunnett’s test).

Article Snippet: Cells cultured for 6 or 24 hours were lysed in M-PER mammalian protein extraction reagent and protease inhibitor cocktail (Thermo Scientific, Waltham MA, USA), then Sema3A levels in the cell lysate were measured using a Human Sema3A ELISA kit (Elabscience, Houston, TX, USA).

Techniques: Incubation, Concentration Assay, Expressing, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Extract of Scutellaria baicalensis induces semaphorin 3A production in human epidermal keratinocytes

doi: 10.1371/journal.pone.0250663

Figure Lengend Snippet: (A) NHEKs were incubated with five plant extracts, samples No. 2, 7, 10, 13, and 76 at a final concentration of 50 μg/mL for 3, 6, and 24 hours at 37°C. The culture supernatants were collected, and then the concentrations of Sema3A protein were measured using ELISA. * p < 0.05, (one-way ANOVA followed by Dunnett’s test). (B) NHEKs were incubated with sample No. 2 ( A . montana ) or No. 13 ( S . baicalensis ) at 50 μg/mL for 6 and 24 hours. After that, the cell lysates were prepared with lysis buffer, and then the levels of Sema3A protein in the cell lysates were measured using ELISA. (C) Cell viability of cultured NHEKs treated with the extract of A . montana or S . baicalensis was measured using a cell counting assay. All results are expressed as means ± S.D. of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (two-way ANOVA followed by Dunnett’s test).

Article Snippet: Cells cultured for 6 or 24 hours were lysed in M-PER mammalian protein extraction reagent and protease inhibitor cocktail (Thermo Scientific, Waltham MA, USA), then Sema3A levels in the cell lysate were measured using a Human Sema3A ELISA kit (Elabscience, Houston, TX, USA).

Techniques: Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Lysis, Cell Culture, Cell Counting

Journal: PLoS ONE

Article Title: Extract of Scutellaria baicalensis induces semaphorin 3A production in human epidermal keratinocytes

doi: 10.1371/journal.pone.0250663

Figure Lengend Snippet: A 100 μL sample of the extract of S . baicalensis , A . montana (100 or 500 μg/mL) or PBS (vehicle) was applied from the stratum corneum side of cultured RHE samples, and the RHE samples were cultured for a further 24 hours. The culture supernatants were collected, and then the concentrations of Sema3A protein were measured using ELISA. All results are expressed as means ± S.D. of three independent experiments. * p < 0.05, (one-way ANOVA followed by Dunnett’s test).

Article Snippet: Cells cultured for 6 or 24 hours were lysed in M-PER mammalian protein extraction reagent and protease inhibitor cocktail (Thermo Scientific, Waltham MA, USA), then Sema3A levels in the cell lysate were measured using a Human Sema3A ELISA kit (Elabscience, Houston, TX, USA).

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Extract of Scutellaria baicalensis induces semaphorin 3A production in human epidermal keratinocytes

doi: 10.1371/journal.pone.0250663

Figure Lengend Snippet: (A) NHEKs were transfected with pGL3- Sema3A vector. After transfection, the cells were incubated with S . baicalensis extract and S . baicalensis -derived compounds: apigenin, baicalein, baicalin, chrysin, and wogonin, at the indicated concentrations, a 3-fold dilution with a maximum concentration of 5 μg/mL, for 24 hours at 37°C. Cell lysates were subjected to the measurement of luciferase activities as described in the Materials and methods. All results are expressed as means ± S.D. of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (one-way ANOVA followed by Dunnett’s test). (B) Chemical formulae of major bioactive flavones from S . baicalensis .

Article Snippet: Cells cultured for 6 or 24 hours were lysed in M-PER mammalian protein extraction reagent and protease inhibitor cocktail (Thermo Scientific, Waltham MA, USA), then Sema3A levels in the cell lysate were measured using a Human Sema3A ELISA kit (Elabscience, Houston, TX, USA).

Techniques: Transfection, Plasmid Preparation, Incubation, Derivative Assay, Concentration Assay, Luciferase

Journal: Oncotarget

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

doi: 10.18632/oncotarget.6831

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

Article Snippet: For SEMA3A protein treatment, cells at 70–80% confluence were treated with varies concentrations of recombinant human Semaphorin3A protein (PEPROTECH, USA) for 48 h. The SEMA3A over-expression vector pCMV-SEMA3A-MCS-EGFP encoding full-length human SEMA3A cDNA and the negative control vector pCMV-MCS-EGFP were subcloned into pAdG135 to generate pAdCMV-SEMA3A-MCS-EGFP and pAdCMV-MCS-EFGP.

Techniques: Immunohistochemistry, Incubation, Negative Control, Expressing

Journal: Oncotarget

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

doi: 10.18632/oncotarget.6831

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

Article Snippet: For SEMA3A protein treatment, cells at 70–80% confluence were treated with varies concentrations of recombinant human Semaphorin3A protein (PEPROTECH, USA) for 48 h. The SEMA3A over-expression vector pCMV-SEMA3A-MCS-EGFP encoding full-length human SEMA3A cDNA and the negative control vector pCMV-MCS-EGFP were subcloned into pAdG135 to generate pAdCMV-SEMA3A-MCS-EGFP and pAdCMV-MCS-EFGP.

Techniques: Expressing

Journal: Oncotarget

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

doi: 10.18632/oncotarget.6831

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

Article Snippet: For SEMA3A protein treatment, cells at 70–80% confluence were treated with varies concentrations of recombinant human Semaphorin3A protein (PEPROTECH, USA) for 48 h. The SEMA3A over-expression vector pCMV-SEMA3A-MCS-EGFP encoding full-length human SEMA3A cDNA and the negative control vector pCMV-MCS-EGFP were subcloned into pAdG135 to generate pAdCMV-SEMA3A-MCS-EGFP and pAdCMV-MCS-EFGP.

Techniques: Expressing

Journal: Oncotarget

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

doi: 10.18632/oncotarget.6831

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

Article Snippet: For SEMA3A protein treatment, cells at 70–80% confluence were treated with varies concentrations of recombinant human Semaphorin3A protein (PEPROTECH, USA) for 48 h. The SEMA3A over-expression vector pCMV-SEMA3A-MCS-EGFP encoding full-length human SEMA3A cDNA and the negative control vector pCMV-MCS-EGFP were subcloned into pAdG135 to generate pAdCMV-SEMA3A-MCS-EGFP and pAdCMV-MCS-EFGP.

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

Journal: Oncotarget

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

doi: 10.18632/oncotarget.6831

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

Article Snippet: For SEMA3A protein treatment, cells at 70–80% confluence were treated with varies concentrations of recombinant human Semaphorin3A protein (PEPROTECH, USA) for 48 h. The SEMA3A over-expression vector pCMV-SEMA3A-MCS-EGFP encoding full-length human SEMA3A cDNA and the negative control vector pCMV-MCS-EGFP were subcloned into pAdG135 to generate pAdCMV-SEMA3A-MCS-EGFP and pAdCMV-MCS-EFGP.

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

Journal: Oncotarget

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

doi: 10.18632/oncotarget.6831

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

Article Snippet: For SEMA3A protein treatment, cells at 70–80% confluence were treated with varies concentrations of recombinant human Semaphorin3A protein (PEPROTECH, USA) for 48 h. The SEMA3A over-expression vector pCMV-SEMA3A-MCS-EGFP encoding full-length human SEMA3A cDNA and the negative control vector pCMV-MCS-EGFP were subcloned into pAdG135 to generate pAdCMV-SEMA3A-MCS-EGFP and pAdCMV-MCS-EFGP.

Techniques: Injection, Control, Isolation

Journal: Oncotarget

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

doi: 10.18632/oncotarget.6831

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

Article Snippet: For SEMA3A protein treatment, cells at 70–80% confluence were treated with varies concentrations of recombinant human Semaphorin3A protein (PEPROTECH, USA) for 48 h. The SEMA3A over-expression vector pCMV-SEMA3A-MCS-EGFP encoding full-length human SEMA3A cDNA and the negative control vector pCMV-MCS-EGFP were subcloned into pAdG135 to generate pAdCMV-SEMA3A-MCS-EGFP and pAdCMV-MCS-EFGP.

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

Journal: Oncotarget

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

doi: 10.18632/oncotarget.6831

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

Article Snippet: For SEMA3A protein treatment, cells at 70–80% confluence were treated with varies concentrations of recombinant human Semaphorin3A protein (PEPROTECH, USA) for 48 h. The SEMA3A over-expression vector pCMV-SEMA3A-MCS-EGFP encoding full-length human SEMA3A cDNA and the negative control vector pCMV-MCS-EGFP were subcloned into pAdG135 to generate pAdCMV-SEMA3A-MCS-EGFP and pAdCMV-MCS-EFGP.

Techniques: Quantitative RT-PCR, Expressing, Western Blot

Journal: Oncotarget

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

doi: 10.18632/oncotarget.6831

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

Article Snippet: For SEMA3A protein treatment, cells at 70–80% confluence were treated with varies concentrations of recombinant human Semaphorin3A protein (PEPROTECH, USA) for 48 h. The SEMA3A over-expression vector pCMV-SEMA3A-MCS-EGFP encoding full-length human SEMA3A cDNA and the negative control vector pCMV-MCS-EGFP were subcloned into pAdG135 to generate pAdCMV-SEMA3A-MCS-EGFP and pAdCMV-MCS-EFGP.

Techniques: Expressing

Journal: PLoS ONE

Article Title: Extract of Scutellaria baicalensis induces semaphorin 3A production in human epidermal keratinocytes

doi: 10.1371/journal.pone.0250663

Figure Lengend Snippet: A list of herbal plants used in screening of Sema3A inducers.

Article Snippet: The culture supernatants were collected after 3, 6, or 24 hours and Sema3A protein concentration was measured using an enzyme-linked immunosorbent assay (ELISA) kit for Sema3A (Uscn Life Science, Wuhan, China), in accordance with the manufacturer’s protocol.

Techniques:

Journal: PLoS ONE

Article Title: Extract of Scutellaria baicalensis induces semaphorin 3A production in human epidermal keratinocytes

doi: 10.1371/journal.pone.0250663

Figure Lengend Snippet: A summary of Sema3A reporter gene activity by herbal plant extracts.

Article Snippet: The culture supernatants were collected after 3, 6, or 24 hours and Sema3A protein concentration was measured using an enzyme-linked immunosorbent assay (ELISA) kit for Sema3A (Uscn Life Science, Wuhan, China), in accordance with the manufacturer’s protocol.

Techniques: Activity Assay

Journal: PLoS ONE

Article Title: Extract of Scutellaria baicalensis induces semaphorin 3A production in human epidermal keratinocytes

doi: 10.1371/journal.pone.0250663

Figure Lengend Snippet: NHEKs were transfected with pGL3- Sema3A . After transfection, the cells were incubated with five plant extracts, namely No. 2, 7, 10, 13, and 76 at a final concentration of 5, 15, and 50 μg/mL for 48 hours at 37°C. Cell lysates were subjected to luciferase activity measurement as described in Materials and methods. All results are expressed as mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (one-way ANOVA followed by Dunnett’s test).

Article Snippet: The culture supernatants were collected after 3, 6, or 24 hours and Sema3A protein concentration was measured using an enzyme-linked immunosorbent assay (ELISA) kit for Sema3A (Uscn Life Science, Wuhan, China), in accordance with the manufacturer’s protocol.

Techniques: Transfection, Incubation, Concentration Assay, Luciferase, Activity Assay

Journal: PLoS ONE

Article Title: Extract of Scutellaria baicalensis induces semaphorin 3A production in human epidermal keratinocytes

doi: 10.1371/journal.pone.0250663

Figure Lengend Snippet: NHEKs were incubated with five plant extracts, samples No. 2, 7, 10, 13, and 76 at a final concentration of 50 μg/mL for 3 and 6 hours at 37°C. Expression of Sema3A mRNA was examined using quantitative real-time PCR analysis. The levels of Sema3A mRNA were normalized relative to those of RPS18 . All results are expressed as mean ± S.D. of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (one-way ANOVA followed by Dunnett’s test).

Article Snippet: The culture supernatants were collected after 3, 6, or 24 hours and Sema3A protein concentration was measured using an enzyme-linked immunosorbent assay (ELISA) kit for Sema3A (Uscn Life Science, Wuhan, China), in accordance with the manufacturer’s protocol.

Techniques: Incubation, Concentration Assay, Expressing, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Extract of Scutellaria baicalensis induces semaphorin 3A production in human epidermal keratinocytes

doi: 10.1371/journal.pone.0250663

Figure Lengend Snippet: (A) NHEKs were incubated with five plant extracts, samples No. 2, 7, 10, 13, and 76 at a final concentration of 50 μg/mL for 3, 6, and 24 hours at 37°C. The culture supernatants were collected, and then the concentrations of Sema3A protein were measured using ELISA. * p < 0.05, (one-way ANOVA followed by Dunnett’s test). (B) NHEKs were incubated with sample No. 2 ( A . montana ) or No. 13 ( S . baicalensis ) at 50 μg/mL for 6 and 24 hours. After that, the cell lysates were prepared with lysis buffer, and then the levels of Sema3A protein in the cell lysates were measured using ELISA. (C) Cell viability of cultured NHEKs treated with the extract of A . montana or S . baicalensis was measured using a cell counting assay. All results are expressed as means ± S.D. of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (two-way ANOVA followed by Dunnett’s test).

Article Snippet: The culture supernatants were collected after 3, 6, or 24 hours and Sema3A protein concentration was measured using an enzyme-linked immunosorbent assay (ELISA) kit for Sema3A (Uscn Life Science, Wuhan, China), in accordance with the manufacturer’s protocol.

Techniques: Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Lysis, Cell Culture, Cell Counting

Journal: PLoS ONE

Article Title: Extract of Scutellaria baicalensis induces semaphorin 3A production in human epidermal keratinocytes

doi: 10.1371/journal.pone.0250663

Figure Lengend Snippet: A 100 μL sample of the extract of S . baicalensis , A . montana (100 or 500 μg/mL) or PBS (vehicle) was applied from the stratum corneum side of cultured RHE samples, and the RHE samples were cultured for a further 24 hours. The culture supernatants were collected, and then the concentrations of Sema3A protein were measured using ELISA. All results are expressed as means ± S.D. of three independent experiments. * p < 0.05, (one-way ANOVA followed by Dunnett’s test).

Article Snippet: The culture supernatants were collected after 3, 6, or 24 hours and Sema3A protein concentration was measured using an enzyme-linked immunosorbent assay (ELISA) kit for Sema3A (Uscn Life Science, Wuhan, China), in accordance with the manufacturer’s protocol.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Extract of Scutellaria baicalensis induces semaphorin 3A production in human epidermal keratinocytes

doi: 10.1371/journal.pone.0250663

Figure Lengend Snippet: (A) NHEKs were transfected with pGL3- Sema3A vector. After transfection, the cells were incubated with S . baicalensis extract and S . baicalensis -derived compounds: apigenin, baicalein, baicalin, chrysin, and wogonin, at the indicated concentrations, a 3-fold dilution with a maximum concentration of 5 μg/mL, for 24 hours at 37°C. Cell lysates were subjected to the measurement of luciferase activities as described in the Materials and methods. All results are expressed as means ± S.D. of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (one-way ANOVA followed by Dunnett’s test). (B) Chemical formulae of major bioactive flavones from S . baicalensis .

Article Snippet: The culture supernatants were collected after 3, 6, or 24 hours and Sema3A protein concentration was measured using an enzyme-linked immunosorbent assay (ELISA) kit for Sema3A (Uscn Life Science, Wuhan, China), in accordance with the manufacturer’s protocol.

Techniques: Transfection, Plasmid Preparation, Incubation, Derivative Assay, Concentration Assay, Luciferase