Journal: American Journal of Cancer Research

Article Title: Pancreatic cancer cell-derived semaphorin 3A promotes neuron recruitment to accelerate tumor growth and dissemination

doi:

Figure Lengend Snippet: SEMA3A and its receptors predict poor prognosis in patients with PDAC. (A) 2933 secretory factors were filtered based on whether they are vesicle proteins and neural regulation-related proteins, and SEMA3A was selected as a candidate factor. (B) Prognostic abilities of SEMA3A and its receptors (C) NRP2 and (D) PLXNA1 were analyzed using PROGgene. (E, F) In addition, prognostic ability of SEMA3A in our own dataset was analyzed.

Article Snippet: The antibodies against SEMA3A (cat No. GTX130671), phospho-ERK (cat No. GTX24819), ERK (cat No. GTX134462), and GAPDH (cat No. GTX627408) were obtained from GeneTex.

Techniques:

Journal: American Journal of Cancer Research

Article Title: Pancreatic cancer cell-derived semaphorin 3A promotes neuron recruitment to accelerate tumor growth and dissemination

doi:

Figure Lengend Snippet: Gene signatures positively correlated with SEMA3A expression in PDAC. SEMA3A coexpression signature in TCGA PAAD was extracted from cBioPortal and analyzed with NetworkAnalyst in KEGG, Reactome, Gene Ontology: Biological Pathway (GO: BP) datasets for enriched pathways and statistical significances. A. Enrichment of axon guidance pathway in KEGG, P<0.001; B. Enrichment of axon guidance pathway in Reactome, P<0.001; C. Enrichment of MAPK signaling pathway in KEGG, P<0.001; D. Enrichment of signaling to ERKs in Reactome, P<0.001; E. Enrichment of actin cytoskeleton organization in GO: BP, P<0.001; F. Enrichment of actin filament organization in GO: BP, P<0.001.

Article Snippet: The antibodies against SEMA3A (cat No. GTX130671), phospho-ERK (cat No. GTX24819), ERK (cat No. GTX134462), and GAPDH (cat No. GTX627408) were obtained from GeneTex.

Techniques: Expressing

Journal: American Journal of Cancer Research

Article Title: Pancreatic cancer cell-derived semaphorin 3A promotes neuron recruitment to accelerate tumor growth and dissemination

doi:

Figure Lengend Snippet: Pancreatic cancer cells express SEMA3A and increase neural migration and neurite outgrowth. Control medium and PDAC-conditioned medium from (A) MIA PaCa-2 or (B) KPC were subjected to Western blotting to detect SEMA3A level. The effect of PDAC-conditioned medium on (C, D) proliferation, (E, F) migration, and (G, H) neurite outgrowth in neuronal cells was analyzed. Green fluorescence in (G) and (H) indicates TUBB3 staining. For differentiated SH-SY5Y cells, control condition is 10% FBS DMEM (MIA PaCa-2 culture medium), and CM condition is MIA PaCa-2-conditioned 10% FBS DMEM. For differentiated Neuro-2a cells, control condition is 10% FBS RPMI (KPC culture medium), and CM condition is KPC-conditioned 10% FBS RPMI. *, P<0.05; **, P<0.01. Scale bar, 5 μm. Error bars represent standard error of the mean (SEM).

Article Snippet: The antibodies against SEMA3A (cat No. GTX130671), phospho-ERK (cat No. GTX24819), ERK (cat No. GTX134462), and GAPDH (cat No. GTX627408) were obtained from GeneTex.

Techniques: Migration, Control, Western Blot, Fluorescence, Staining

Journal: American Journal of Cancer Research

Article Title: Pancreatic cancer cell-derived semaphorin 3A promotes neuron recruitment to accelerate tumor growth and dissemination

doi:

Figure Lengend Snippet: SEMA3A blockage decreases PDAC-induced migration and recruitment of neuronal cells. Control IgG or SEMA3A antibody was added into the conditioned medium from MIA PaCa-2, in combination with vehicle or SEMA3A peptide, and used to treat differentiated SH-SY5Y. Subsequent effects of these combinations on (A) migration, (B) neurite outgrowth, and (C) 3D recruitment were analyzed. (C) MIA PaCa-2 cells were labeled with the green fluorescence membrane dye, DiO, and differentiated SH-SY5Y cells were labeled with the red fluorescence membrane dye, Dil. (D) The expression of SEMA3A in mouse KPC was inhibited using shRNA. Conditioned media from control- or SEMA3A-depleted KPCs were used to treat differentiated Neuro-2a cells and the effects on (D) migration, (E) neurite outgrowth and (F) 3D recruitment were assayed. *, P<0.05; **, P<0.01; ***, P<0.001. Scale bar, 100 μm. Error bars represent SEM.

Article Snippet: The antibodies against SEMA3A (cat No. GTX130671), phospho-ERK (cat No. GTX24819), ERK (cat No. GTX134462), and GAPDH (cat No. GTX627408) were obtained from GeneTex.

Techniques: Migration, Control, Labeling, Fluorescence, Membrane, Expressing, shRNA

Journal: American Journal of Cancer Research

Article Title: Pancreatic cancer cell-derived semaphorin 3A promotes neuron recruitment to accelerate tumor growth and dissemination

doi:

Figure Lengend Snippet: Inhibition of PLXNA1 and NRP2 on neuronal cells reduces SEMA3A-induced migration, neurite outgrowth, and neural recruitment. (A) Protein level of PLXNA1 and NRP2 on the surface of differentiated SH-SY5Y was determined using flow cytometry [red, unstain; blue, 2’Ab only; orange, α-PLXNA1 (left) or α-NRP2 (right)]. Differentiated SH-SY5Y was treated with control IgG, α-PLXNA1, or α-NRP2, and the effects of these treatments on (B) migration, (C) neurite outgrowth, and (D) 3D recruitment were analyzed. (E) Expression of PLXNA1 in differentiated Neuro-2a cells was inhibited using shRNA and the level of PLXNA1 was determined using RT-PCR. The effect of PLXNA1 depletion on KPC-conditioned medium-induced (F) migration, (G) neurite outgrowth, and (H) 3D recruitment of differentiated Neuro-2a cells was analyzed. (I) Expression of NRP2 on the surface of differentiated Neuro-2a cells was assayed using flow cytometry (red, unstain; blue, 2’Ab only; orange, α-NRP2). The effect of NRP2 inhibition through neutralizing antibody on KPC-conditioned medium-induced (J) migration, (K) neurite outgrowth, and (L) 3D recruitment of differentiated Neuro-2a cells was analyzed. *, P<0.05; **, P<0.01; ***, P<0.001. Error bars represent SEM.

Article Snippet: The antibodies against SEMA3A (cat No. GTX130671), phospho-ERK (cat No. GTX24819), ERK (cat No. GTX134462), and GAPDH (cat No. GTX627408) were obtained from GeneTex.

Techniques: Inhibition, Migration, Flow Cytometry, Control, Expressing, shRNA, Reverse Transcription Polymerase Chain Reaction

Journal: American Journal of Cancer Research

Article Title: Pancreatic cancer cell-derived semaphorin 3A promotes neuron recruitment to accelerate tumor growth and dissemination

doi:

Figure Lengend Snippet: SEMA3A activates MEK in neuronal cells to promote their motility. Differentiated SH-SY5Y and Neuro-2a cells were treated with conditioned medium of MIA PaCa-2 or KPC in the presence or absence of the MEK inhibitor, trametinib. The effect of conditioned medium on ERK phosphorylation in (A) differentiated SH-SY5Y cells and (B) differentiated Neuro-2a cells was investigated using Western blotting. In addition, the effect of trametinib on conditioned medium-induced (C, D) migration, (E, F) neurite outgrowth, and (G, H) 3D recruitment was analyzed. *, P<0.05; **, P<0.01. Error bars represent SEM.

Article Snippet: The antibodies against SEMA3A (cat No. GTX130671), phospho-ERK (cat No. GTX24819), ERK (cat No. GTX134462), and GAPDH (cat No. GTX627408) were obtained from GeneTex.

Techniques: Phospho-proteomics, Western Blot, Migration

Journal: American Journal of Cancer Research

Article Title: Pancreatic cancer cell-derived semaphorin 3A promotes neuron recruitment to accelerate tumor growth and dissemination

doi:

Figure Lengend Snippet: Inhibition of SEMA3A reduces pancreatic tumor growth, nerve innervation, and metastasis in an orthotopic mouse model. Control shLuc- or shSEMA3A-KPC cells (2 × 105) were orthotopically injected into the pancreas of C57BL/6 mice. Three weeks post-inoculation the mice were sacrificed and tissues were harvested. (A, B) Tumor size and expressions of (C) SEMA3A, (D) PCNA, (E) TUBB3, and (F) phospho-ERK were compared between control and SEMA3A-depleted groups. In addition, the (G) number and (H) size of the metastatic nodes found in the abdomen were compared. *, P<0.05; **, P<0.01; ***, P<0.001. Scale bar, 50 μm. MFI, mean fluorescence intensity. Error bars represent SEM.

Article Snippet: The antibodies against SEMA3A (cat No. GTX130671), phospho-ERK (cat No. GTX24819), ERK (cat No. GTX134462), and GAPDH (cat No. GTX627408) were obtained from GeneTex.

Techniques: Inhibition, Control, Injection, Fluorescence

Journal: PLoS ONE

Article Title: Mutational Spectrum of Semaphorin 3A and Semaphorin 3D Genes in Spanish Hirschsprung patients

doi: 10.1371/journal.pone.0054800

Figure Lengend Snippet: SEMA3A sequence variants detected in the current study.

Article Snippet: Sections were incubated at 4°C overnight with the primary antibodies anti- SEMA3A (1∶50 dilution, anti-rabbit, AntibodyBcn, Barcelona, Spain) and anti- SEMA3D (1∶5 dilution, anti-rabbit, Novus Biologicals, Cambridge, UK).

Techniques: Sequencing

Journal: PLoS ONE

Article Title: Mutational Spectrum of Semaphorin 3A and Semaphorin 3D Genes in Spanish Hirschsprung patients

doi: 10.1371/journal.pone.0054800

Figure Lengend Snippet: SEMA3A and SEMA3D missense variants detected in isolated HSCR patients.

Article Snippet: Sections were incubated at 4°C overnight with the primary antibodies anti- SEMA3A (1∶50 dilution, anti-rabbit, AntibodyBcn, Barcelona, Spain) and anti- SEMA3D (1∶5 dilution, anti-rabbit, Novus Biologicals, Cambridge, UK).

Techniques: Isolation, Mutagenesis

Journal: PLoS ONE

Article Title: Mutational Spectrum of Semaphorin 3A and Semaphorin 3D Genes in Spanish Hirschsprung patients

doi: 10.1371/journal.pone.0054800

Figure Lengend Snippet: The SEMA3A staining illustrated that the expression was present at smooth muscle (D, E, F) and submucous (G, H, I) layers, as well as in myenteric (J, K) and submucous plexuses (M, N) either in normal colon (A, D, G, J, M) and patients with A131T-3A (B, E, H, K, N) and S598G-3A mutations (C, F, I). The FFPE tissue block from patient with S598G had no all tissue layers. Scale bars: A–C = 200 µm and the rest of pictures = 10 µm.

Article Snippet: Sections were incubated at 4°C overnight with the primary antibodies anti- SEMA3A (1∶50 dilution, anti-rabbit, AntibodyBcn, Barcelona, Spain) and anti- SEMA3D (1∶5 dilution, anti-rabbit, Novus Biologicals, Cambridge, UK).

Techniques: Staining, Expressing, Blocking Assay

Journal: Biochemical and biophysical research communications

Article Title: Semaphorin 3A potentiates the profibrotic effects of transforming growth factor-β1 in the cornea

doi: 10.1016/j.bbrc.2019.10.107

Figure Lengend Snippet: A. Unoperated, cornea triple labelled for SEMA3A, α-SMA and DAPI. The epithelium is uppermost. Individual stains (monochrome) are shown in B-D. Keratocytes (arrowed) are faintly SEMA3A positive. Note total absence of α-SMA staining. E. Photograph of a central cat cornea 4 weeks after PRK, triple labelled as in A. Individual stains are shown in F-H. Arrows indicate α-SMA-negative cells that are strongly SEMA3A-positive. Note the zone of cellular hyper-density below the α-SMA-positive layer (demarcated with yellow line), where a significant proportion of cells [putative fibroblasts] are SEMA3A-positive.

Article Snippet: Sections in the ablation center (or the geometric center of unoperated corneas) were selected for immunohistochemistry; they were co-incubated with a primary mouse monoclonal anti-α-SMA (1:100; Thermo Fisher Scientific, Waltham, MA, USA) and a feline-specific, rabbit polyclonal anti-SEMA3A antibody (1:100; YenZym Antibodies LLC., South San Francisco, CA, USA) overnight at 4°C.

Techniques: Staining

Journal: Biochemical and biophysical research communications

Article Title: Semaphorin 3A potentiates the profibrotic effects of transforming growth factor-β1 in the cornea

doi: 10.1016/j.bbrc.2019.10.107

Figure Lengend Snippet: Representative Western blot showing similar, relative SEMA3A expression levels in whole cell lysates and concentrated supernatants (S/N) of cat corneal fibroblasts (F) and myofibroblasts (M). Expression of α-SMA was used as an indicator of myofibroblast differentiation in the cell lysates.

Article Snippet: Sections in the ablation center (or the geometric center of unoperated corneas) were selected for immunohistochemistry; they were co-incubated with a primary mouse monoclonal anti-α-SMA (1:100; Thermo Fisher Scientific, Waltham, MA, USA) and a feline-specific, rabbit polyclonal anti-SEMA3A antibody (1:100; YenZym Antibodies LLC., South San Francisco, CA, USA) overnight at 4°C.

Techniques: Western Blot, Expressing

Journal: Biochemical and biophysical research communications

Article Title: Semaphorin 3A potentiates the profibrotic effects of transforming growth factor-β1 in the cornea

doi: 10.1016/j.bbrc.2019.10.107

Figure Lengend Snippet: A. Representative Western blot showing protein levels for COL1, t-FN, EDA-FN and α-SMA in cells treated with recombinant human SEMA3A with/without TGF-β1. Adding SEMA3A potentiated TGF-β1-induced increases in EDA-FN, t-FN, COL1 and α-SMA relative to TGF-β1 alone. B. Plot of relative densitometry data for EDA-FN, t-FN, COL1 and α-SMA with respect to β-actin. Data are means±SEM over 4 experiments. **ANOVA relative to baseline, p<0.0005. *ANOVA between 2 treatment groups, p<0.05. C. Representative Western blot showing SEMA3A levels in cells transfected with Sema3A siRNA or control (non-targeting) siRNA. D. Representative Western blot showing relative levels of COL1, t-FN, EDA-FN and α-SMA in fibroblasts transfected with control siRNA or Sema3A siRNA and cultured with/without TGF-β1. E. Plot of relative densitometry data for EDA-FN, t-FN, COL1 and α-SMA with respect to β-actin. Data are means±SEM over 3 experiments. *ANOVA relative to baseline, p<0.05. ns ANOVA between 2 treatment groups, p=0.227.

Article Snippet: Sections in the ablation center (or the geometric center of unoperated corneas) were selected for immunohistochemistry; they were co-incubated with a primary mouse monoclonal anti-α-SMA (1:100; Thermo Fisher Scientific, Waltham, MA, USA) and a feline-specific, rabbit polyclonal anti-SEMA3A antibody (1:100; YenZym Antibodies LLC., South San Francisco, CA, USA) overnight at 4°C.

Techniques: Western Blot, Recombinant, Transfection, Control, Cell Culture

Journal: Biochemical and biophysical research communications

Article Title: Semaphorin 3A potentiates the profibrotic effects of transforming growth factor-β1 in the cornea

doi: 10.1016/j.bbrc.2019.10.107

Figure Lengend Snippet: A. Phase contrast images (top row) show a change from fibroblastic to myofibroblastic morphologies in both cells transfected with control and Sema3A siRNA, but α-SMA staining (green in bottom row) is decreased in Sema3A siRNA-treated cells. B. Phalloidin staining of different regions on the same plates as in A shows persistence of F-actin fibers and clear morphological characteristics of larger myofibroblasts in the two plates treated with TGF-β1, even when α-SMA expression was decreased, as in the Sema3A siRNA+TGF-β1 condition.

Article Snippet: Sections in the ablation center (or the geometric center of unoperated corneas) were selected for immunohistochemistry; they were co-incubated with a primary mouse monoclonal anti-α-SMA (1:100; Thermo Fisher Scientific, Waltham, MA, USA) and a feline-specific, rabbit polyclonal anti-SEMA3A antibody (1:100; YenZym Antibodies LLC., South San Francisco, CA, USA) overnight at 4°C.

Techniques: Transfection, Control, Staining, Expressing