Journal: Journal of Cellular Physiology

Article Title: Maintenance of Bone Homeostasis by DLL1‐Mediated Notch Signaling

doi: 10.1002/jcp.25647

Figure Lengend Snippet: Effects of osteoblast‐specific DLL1 expression in osteoclast differentiation in vitro. (a) The numbers of TRAP(+) osteoclasts formed in cytokine‐induced culture, (b) mixed culture, and (c) osteoblast‐osteoclast co‐culture in vitro. (a and b) Representative data from six independent experiments are shown. (c) Representative data from three independent experiments using calvarial osteoblasts obtained from a total of three non‐transgenic and six DLL1 littermates. Ob, osteoblasts. Experiments were performed at least in triplicate wells. Error bars, mean ± SD; * P < 0.05. Quantitative PCR analyses of Rankl (d) and Semaphorin 3B (f) expression in non‐transgenic and DLL1 osteoclast culture. Representative data from five independent experiments are shown. Analyses were performed at least in triplicate wells. Error bars, mean ± SD; * P < 0.05. (e) Immunostaining of 4‐week‐old mice. BM specimens were stained for RANKL and then for TRAP. Arrowheads indicate RANKL‐positive, bone‐lining, and matrix‐embedded osteoblasts. Four independent littermate pairs at the ages of 2–4 weeks were stained.

Article Snippet: Jagged2: Mm01325629_m1, Hes1: Mm01342805_m1, Hey1: Mm00468865_m1, Col1a1: mm0080166_g1, ALP: Mm00475834_m1, osteocalcin (bglap3): Mm03413826_m1, Runx2: Mm00501580_m1, osterix (sp7): Mm04209856_m1, RANKL (tnfsf11): Mm00441906_m1, Sema3b: Mm00436477_m1.

Techniques: Expressing, In Vitro, Co-Culture Assay, Transgenic Assay, Real-time Polymerase Chain Reaction, Immunostaining, Staining

Journal: Oncogene

Article Title: GATA3 Targets Semaphorin 3B in Mammary Epithelial Cells to Suppress Breast Cancer Progression and Metastasis

doi: 10.1038/onc.2017.165

Figure Lengend Snippet: In silico analysis of breast cancer databases. (a) Analysis of TCGA database. Heat map indicating gene expression analysis for GATA3 , SEMA3B and MYC in multiple breast cancer samples. (N = 597) (b) Analysis of TCGA database via Regulome explorer. Data present a collection of proteins including GATA3 that may reside in the same protein network as SEMA3B. (c) Analysis of SEMA3B expression via the GSE9014 database. SEMA3B expression is significantly lowered in the invasive breast carcinoma samples. (Normal: N = 6, Invasive breast carcinoma: N = 53, P = 1.03E-21) (d) TCGA analysis indicating significantly lowered SEMA3B expression in triple negative breast cancer samples. (Normal: N = 244, Triple negative: N = 49, P = 4.06E-9) (e) Analysis of SEMA3B expression in PAM50 subtypes using the METABRIC database. Luminal breast cancers possess higher levels of SEMA3B expression than the basal subtype, P = <0.0001, Normal: N = 202, Luminal A: N = 721, Luminal B: N = 492. HER2: N = 240, Basal: N = 331) (f) Analysis of breast cancer patient survival . Tumor samples exhibiting lower SEMA3B expression show poorer patient prognosis. (Concordance index = 62.3, Log-rank equal curves P = 1.6E-4, R^ 2 = 0.005/0.941, Risk groups hazard ratio = 2.4, P = 2.61E-6)

Article Snippet: SEMA3B siRNA (Santa Cruz Biotechnology INC.).

Techniques: In Silico, Gene Expression, Expressing

Journal: Oncogene

Article Title: GATA3 Targets Semaphorin 3B in Mammary Epithelial Cells to Suppress Breast Cancer Progression and Metastasis

doi: 10.1038/onc.2017.165

Figure Lengend Snippet: GATA3 directly controls SEMA3B expression. (a) ChIP analysis indicating GATA3 transcription factor binding to SEMA3B promoter in EpH4.9, T47D and MDA-MB-231 cells. (b) qPCR analysis of GATA3 and SEMA3B expression in MDA-MB-231 cells, N = 6. Overexpression of GATA3 in MDA-MB-231 cells results in elevation of SEMA3B levels. (c) Western blot analysis of MDA-MB-231 cell lysate. Overexpression of GATA3 enhances SEMA3B expression. (d) Immunostaining of MDA-MB-231 (control) and GATA3 overexpressing MDA-MB-231 cells. Overexpression of GATA3 upregulates SEMA3B levels. Bar 50 μm. (e) qPCR analysis of SEMA3B in NMuMG cells, N = 2. Lowering GATA3 expression via siRNA results in downregulation of SEMA3B levels.

Article Snippet: SEMA3B siRNA (Santa Cruz Biotechnology INC.).

Techniques: Expressing, Binding Assay, Over Expression, Western Blot, Immunostaining, Control

Journal: Oncogene

Article Title: GATA3 Targets Semaphorin 3B in Mammary Epithelial Cells to Suppress Breast Cancer Progression and Metastasis

doi: 10.1038/onc.2017.165

Figure Lengend Snippet: Inhibition of cellular migration and proliferation by GATA3 relies on presence of SEMA3B. (a) Bright-field images of MDA-MB-231 stable cell lines in 2D cell cultures. Bar, 50 μm. (b) Phase-contrast images of MDA-MB-231 stable cell lines in 3D cultures. Arrows indicate the presence of invadopodia moving outward from the colony. Bar, 200 μm. (c) Quantification of 3D Matrigel culture colonies. (N = 7) (d) qRT-PCR analysis of EMT-associated genes. RNA samples from MDA-MB-231 control and MDA-SEMA3B cells were collected. qRT-PCR analysis indicates that SEMA3B lowers EMT gene levels. SEMA3B enhanced E-cadherin levels compared to control. (e) Scratch assay analysis indicating difference in cellular migration in MDA-MB-231 stable cell lines. N = 6 (f) Quantification of cell infiltration of gaps between the two invading cell fronts. The gap was measured using ImageJ software. Y-axis indicates experimental group/control group. (N = 6). (g) Colony formation assay. MDA-MB-231 stable cell lines were cultured in 6-well plates to allow colony growth. Colonies were stained with crystal violet and counted. N = 12. (h) Graph representing the quantification of Giemsa stained colonies. (N = 12).

Article Snippet: SEMA3B siRNA (Santa Cruz Biotechnology INC.).

Techniques: Inhibition, Migration, Stable Transfection, Quantitative RT-PCR, Control, Wound Healing Assay, Software, Colony Assay, Cell Culture, Staining

Journal: Oncogene

Article Title: GATA3 Targets Semaphorin 3B in Mammary Epithelial Cells to Suppress Breast Cancer Progression and Metastasis

doi: 10.1038/onc.2017.165

Figure Lengend Snippet: Loss of SEMA3B disrupts GATA3 tumor suppressive activity. (a) MDA-SEMA3B and (b) MDA-GATA3-SEMA3B KD stable cell lines where transplanted in 8-week-old female nude mice via orthotopic injection. Color photographs show a representative set of gross tumors from each transplanted group. Graphs indicates tumor measurement during the course of experiment, N = 10 for each group. (c) Immunostaining and quantification analysis for Ki67 expression in the tumor sections (N = 6). Bar, 100 μm. (d) Analysis of inguinal lymph node weight from transplanted mice, N = 10 for each group. (e) H&E analysis of lymph node sections indicating tumor metastasis. For each transplanted group, metastatic tumor cells to the lymph node was quantified (N = 6). Bar = 200 μm. (f) Western blot analysis indicating phosphorylation status of LIMK1 and LIMK2 in control and MDA-SEMA3B cell extracts. Total LIMK1 and LIMK2 indicate equal loading of samples. Protein bands 72 kDa.

Article Snippet: SEMA3B siRNA (Santa Cruz Biotechnology INC.).

Techniques: Activity Assay, Stable Transfection, Injection, Immunostaining, Expressing, Western Blot, Phospho-proteomics, Control

Journal: Cell

Article Title: Human Semaphorin 3 Variants Link Melanocortin Circuit Development and Energy Balance

doi: 10.1016/j.cell.2018.12.009

Figure Lengend Snippet:

Article Snippet: Human Sema3B expression plasmid , OriGene , CAT#: RC223532.

Techniques: Subcloning, Recombinant, Isolation, Expressing, Plasmid Preparation, Software

Journal: Arthritis & Rheumatology (Hoboken, N.j.)

Article Title: Central Role of Semaphorin 3B in a Serum‐Induced Arthritis Model and Reduced Levels in Patients With Rheumatoid Arthritis

doi: 10.1002/art.42065

Figure Lengend Snippet: Semaphorin 3B (Sema3B) deficiency increases the severity of serum‐induced arthritis. A , Daily global arthritis scores in wild‐type (WT) mice (n = 10) and Sema3B −/− mice (n = 10). Values are the mean ± SEM. B , Inflammation (I) scores, cartilage damage (CD) scores, and bone erosion (BE) scores in mice in each group. C , Representative images of histologic features in the mouse joints, visualized using hematoxylin and eosin (H&E) and toluidine blue staining (n = 10). Synovial cell infiltration ( asterisks ), bone erosion ( black arrow ), and cartilage damage ( white arrows ) are shown. D , Expression of Sema3B mRNA in the joints and fibroblast‐like synoviocytes (FLS) of WT mice (n = 6–8) and Sema3B −/− mice (n = 6–8). E and F , Representative immunoblot ( E ) and densitometric analysis ( F ) of Sema3B expression in FLS from WT mice (n = 4) and Sema3B −/− mice (n = 4). In B , D , and F , symbols represent individual mice; bars show the mean ± SEM. * = P < 0.05; ** = P < 0.01. ### = P < 0.001; #### = P < 0.0001, versus nonarthritic WT control mice.

Article Snippet: Sema3B (Biomatik) levels in the serum of patients with arthralgia and patients with RA, Sema3B (Abbexa) levels in the serum of arthritic mice, and interleukin‐6 (IL‐6) (eBioscience) and tumor necrosis factor (TNF) (R&D Systems) levels in cell‐free assay supernatants of mouse FLS were measured using an enzyme‐linked immunosorbent assay, according to the manufacturer's instructions.

Techniques: Staining, Expressing, Western Blot, Control

Journal: Arthritis & Rheumatology (Hoboken, N.j.)

Article Title: Central Role of Semaphorin 3B in a Serum‐Induced Arthritis Model and Reduced Levels in Patients With Rheumatoid Arthritis

doi: 10.1002/art.42065

Figure Lengend Snippet: Sema3B deficiency enhances the activation of inflammatory pathways. A , Expression of differentially expressed gene (DEG) mRNA in the forepaws of WT or Sema3B −/− mice in a model of rheumatoid arthritis (RA) (n = 5 each) relative to that in nonarthritic control (Ct) mice (n = 4). Data are presented as a heatmap showing the lowest (blue) and highest (orange) mRNA expression levels. B , Gene Ontology analysis of the biologic processes associated with DEGs specific to arthritic WT mice, those specific to arthritic Sema3B −/− mice, or those shared between both groups. C , Expression of mRNA for inflammatory mediators, analyzed by RNA‐Seq in the forepaws of nonarthritic control mice (n = 4) and arthritic WT mice or Sema3B −/− mice (n = 5). Symbols represent individual mice; bars show the mean ± SEM. * = P < 0.05; ** = P < 0.01; *** = P < 0.001; **** = P < 0.0001. # = P < 0.05; ## = P < 0.01; ### = P < 0.001; #### = P < 0.0001, versus nonarthritic WT control mice. LDL = low‐density lipoprotein (see Figure for other definitions). Color figure can be viewed in the online issue, which is available at http://onlinelibrary.wiley.com/doi/10.1002/art.42065/abstract .

Article Snippet: Sema3B (Biomatik) levels in the serum of patients with arthralgia and patients with RA, Sema3B (Abbexa) levels in the serum of arthritic mice, and interleukin‐6 (IL‐6) (eBioscience) and tumor necrosis factor (TNF) (R&D Systems) levels in cell‐free assay supernatants of mouse FLS were measured using an enzyme‐linked immunosorbent assay, according to the manufacturer's instructions.

Techniques: Activation Assay, Expressing, Control, RNA Sequencing

Journal: Arthritis & Rheumatology (Hoboken, N.j.)

Article Title: Central Role of Semaphorin 3B in a Serum‐Induced Arthritis Model and Reduced Levels in Patients With Rheumatoid Arthritis

doi: 10.1002/art.42065

Figure Lengend Snippet: Sema3B deficiency enhances the activation of inflammatory pathways. A , Expression of mRNA for inflammatory mediators analyzed by quantitative PCR (qPCR) of the forepaws in nonarthritic control mice (n = 8) and arthritic WT mice or Sema3B −/− mice (n = 10). B , Densitometric analysis and representative immunoblot of tumor necrosis factor (TNF) in the joints of arthritic WT mice (n = 10) and arthritic Sema3B −/− mice (n = 10). C , Expression of Sema3B mRNA receptors analyzed by qPCR of the forepaws of nonarthritic control mice (n = 8) and arthritic WT mice or Sema3B −/− mice (n = 10 each). D and E , Densitometric analysis and representative immunoblot of neuropilin 1 (NRP‐1) and plexin A2 expression ( D ) and ERK activation ( E ) in the joints of arthritic WT mice (n = 6) and arthritic Sema3B −/− mice (n = 6). Symbols represent individual mice; bars show the mean ± SEM. * = P < 0.05; ** = P < 0.01; *** = P < 0.001; **** = P < 0.0001. # = P < 0.05; ## = P < 0.01; ### = P < 0.001; #### = P < 0.0001, versus nonarthritic WT control mice. Tub = tubulin (see Figure for other definitions). Color figure can be viewed in the online issue, which is available at http://onlinelibrary.wiley.com/doi/10.1002/art.42065/abstract .

Article Snippet: Sema3B (Biomatik) levels in the serum of patients with arthralgia and patients with RA, Sema3B (Abbexa) levels in the serum of arthritic mice, and interleukin‐6 (IL‐6) (eBioscience) and tumor necrosis factor (TNF) (R&D Systems) levels in cell‐free assay supernatants of mouse FLS were measured using an enzyme‐linked immunosorbent assay, according to the manufacturer's instructions.

Techniques: Activation Assay, Expressing, Real-time Polymerase Chain Reaction, Control, Western Blot

Journal: Arthritis & Rheumatology (Hoboken, N.j.)

Article Title: Central Role of Semaphorin 3B in a Serum‐Induced Arthritis Model and Reduced Levels in Patients With Rheumatoid Arthritis

doi: 10.1002/art.42065

Figure Lengend Snippet: Sema3B deficiency enhances activation of inflammatory pathways and the migratory capacity of FLS. A and B , Expression of mRNA for inflammatory mediators ( A ) and tumor necrosis factor (TNF) and interleukin‐6 (IL‐6) protein secretion ( B ) in FLS (at passage 4) from nonarthritic control mice (n = 6) and arthritic WT mice or Sema3B −/− mice (n = 7). C , Migration of FLS (at passage 4) from arthritic WT mice and arthritic Sema3B −/− mice after culture in 1% or 10% fetal bovine serum (FBS) for 24 hours. D , Migration of mouse FLS (at passage 6) from arthritic Sema3B −/− mice stimulated with recombinant mouse Sema3B (rmSema3B) (100 ng/ml) after culture in 1% or 10% FBS for 24 hours. E , Densitometric analysis and representative immunoblot of ERK activation in FLS (at passage 4) from arthritic WT mice (n = 4) and arthritic Sema3B −/− mice (n = 4). In A–C and E , symbols represent individual mice; bars show the mean ± SEM. * = P < 0.05; ** = P < 0.01; *** = P < 0.001; **** = P < 0.0001. # = P < 0.05; ## = P < 0.01; ### = P < 0.001; #### = P < 0.0001, versus nonarthritic WT control mice. See Figure for other definitions. Color figure can be viewed in the online issue, which is available at http://onlinelibrary.wiley.com/doi/10.1002/art.42065/abstract .

Article Snippet: Sema3B (Biomatik) levels in the serum of patients with arthralgia and patients with RA, Sema3B (Abbexa) levels in the serum of arthritic mice, and interleukin‐6 (IL‐6) (eBioscience) and tumor necrosis factor (TNF) (R&D Systems) levels in cell‐free assay supernatants of mouse FLS were measured using an enzyme‐linked immunosorbent assay, according to the manufacturer's instructions.

Techniques: Activation Assay, Expressing, Control, Migration, Recombinant, Western Blot

Journal: Arthritis & Rheumatology (Hoboken, N.j.)

Article Title: Central Role of Semaphorin 3B in a Serum‐Induced Arthritis Model and Reduced Levels in Patients With Rheumatoid Arthritis

doi: 10.1002/art.42065

Figure Lengend Snippet: Sema3B reduces the severity of serum‐induced arthritis. A , Daily global arthritis scores in arthritic WT mice treated on days 0, 2, and 4 with control phosphate buffered saline (PBS) (n = 4), isotype control IgG (10 μg) (n = 6), or recombinant mouse Sema3B (rmSema3B) (10 μg) (n = 6). Values are the mean ± SEM. B , Inflammation scores, cartilage damage scores, and bone erosion scores in mice in each group. C , Representative images of histologic features in the mouse joints visualized with H&E and toluidine blue staining. Synovial cell infiltration ( asterisks ), bone erosion ( black arrows ), and cartilage damage ( white arrows ) are shown. D , Longitudinal serum Sema3B levels in the mouse groups analyzed in A . E , Expression of mRNA for inflammatory mediators in the forepaws of mice analyzed in A . In B and E , symbols represent individual mice; bars show the mean ± SEM. * = P < 0.05; ** = P < 0.01; *** = P < 0.001. # = P < 0.05; ## = P < 0.01, versus Sema3B‐treated mice on day 4. See Figure for other definitions. Color figure can be viewed in the online issue, which is available at http://onlinelibrary.wiley.com/doi/10.1002/art.42065/abstract .

Article Snippet: Sema3B (Biomatik) levels in the serum of patients with arthralgia and patients with RA, Sema3B (Abbexa) levels in the serum of arthritic mice, and interleukin‐6 (IL‐6) (eBioscience) and tumor necrosis factor (TNF) (R&D Systems) levels in cell‐free assay supernatants of mouse FLS were measured using an enzyme‐linked immunosorbent assay, according to the manufacturer's instructions.

Techniques: Control, Saline, Recombinant, Staining, Expressing

Journal: Arthritis & Rheumatology (Hoboken, N.j.)

Article Title: Central Role of Semaphorin 3B in a Serum‐Induced Arthritis Model and Reduced Levels in Patients With Rheumatoid Arthritis

doi: 10.1002/art.42065

Figure Lengend Snippet: Semaphorin 3B (Sema3B) expression is reduced during the progression of rheumatoid arthritis (RA). A and B , Expression of Sema3B mRNA in synovial tissue ( A ) and Sema3B protein in serum ( B ) from patients with arthralgia (n = 8) and those with established RA (n = 10). C , Sema3B levels in a longitudinal cohort of patients with clinically suspect arthralgia (CSA) (n = 40) who had disease that progressed to RA (n = 20) or those whose disease remained as arthralgia after 2 years of follow‐up (n = 20). D , Sema3B levels in a longitudinal cohort of patients with CSA at presentation of arthralgia (n = 8), at presentation of RA (n = 8), and 1 year (n = 8) and 2 years (n = 4) after RA diagnosis. Symbols represent individual patients; bars show the mean ± SEM. * = P < 0.05; ** = P < 0.01; *** = P < 0.001. # = P < 0.05, ### = P < 0.001, versus presentation.

Article Snippet: Sema3B (Biomatik) levels in the serum of patients with arthralgia and patients with RA, Sema3B (Abbexa) levels in the serum of arthritic mice, and interleukin‐6 (IL‐6) (eBioscience) and tumor necrosis factor (TNF) (R&D Systems) levels in cell‐free assay supernatants of mouse FLS were measured using an enzyme‐linked immunosorbent assay, according to the manufacturer's instructions.

Techniques: Expressing, Biomarker Discovery

Journal: Neural Development

Article Title: Boundary cap cells constrain spinal motor neuron somal migration at motor exit points by a semaphorin-plexin mechanism

doi: 10.1186/1749-8104-2-21

Figure Lengend Snippet: Expression of semaphorin genes in the trunk reveals putative repellent ligands are localised to ventral BC cells. (a-f') In situ hybridisations of HH stage 23 chick embryo transverse cryosections (b-f), in parallel with cad7(green) and neurofilament (NF; red) antibody staining on adjacent sections (a,b'-f') show that cad7-positive BC cells at the spinal cord DREZ (red arrows) and MEP (black arrows) express Sema3B (b), Sema3G (e) and Sema6A (f). In contrast, Sema3F (d) is not expressed at the MEP. Prominent expression of Sema3C (c) in motor neurons contrasts with a weak signal in ventral nerve roots. Bar = 100 μm.

Article Snippet: A cDNA probe for chick Plexin-A1 [ ] was a gift from Dr Britta Eickholt; chick Npn-1 from Dr Jon Raper; chick Npn-2 from Dr Gera Neufeld; chick Sema3F from Dr Yuji Watanabe; chick Plexin-A2 (ChEST887n12), Plexin-A4 (ChEST202o14), chick Sema3B (ChEST771a21), chick Sema3C (ChEST997l4), chick Sema6A (ChEST667n18), chick MICAL2 (ChEST58n1), and chick MICAL3 (ChEST59i4) were obtained from the BBSRC chick EST database [ ] via the MRC Geneservice.

Techniques: Expressing, In Situ, Staining

Journal: Neural Development

Article Title: Boundary cap cells constrain spinal motor neuron somal migration at motor exit points by a semaphorin-plexin mechanism

doi: 10.1186/1749-8104-2-21

Figure Lengend Snippet: Genetic ablation of Sema3B in the mouse does not lead to ectopic positioning of spinal motor neurons. (a) As seen in the chick, in the E12.5 mouse trunk, Sema3B is expressed by BC cells located at the spinal cord DREZ (red arrows) and MEP (black arrows). Bar = 100 μm. (b) A quantitative analysis of the incidence of ectopic motor neurons at hindlimb level reveals that there is no significant difference between E12.5 Sema3B null mice and heterozygous and wild-type littermates (n = 3 each). This is in sharp contrast to E12.5 Npn-2 null mice, which is included for comparison (n = 4). ** P ≤ 0.01; two-tailed t -test.

Article Snippet: A cDNA probe for chick Plexin-A1 [ ] was a gift from Dr Britta Eickholt; chick Npn-1 from Dr Jon Raper; chick Npn-2 from Dr Gera Neufeld; chick Sema3F from Dr Yuji Watanabe; chick Plexin-A2 (ChEST887n12), Plexin-A4 (ChEST202o14), chick Sema3B (ChEST771a21), chick Sema3C (ChEST997l4), chick Sema6A (ChEST667n18), chick MICAL2 (ChEST58n1), and chick MICAL3 (ChEST59i4) were obtained from the BBSRC chick EST database [ ] via the MRC Geneservice.

Techniques: Comparison, Two Tailed Test

Journal: Oxidative Medicine and Cellular Longevity

Article Title: The lncRNA SEMA3B-AS1/HMGB1/FBXW7 Axis Mediates the Peritoneal Metastasis of Gastric Cancer by Regulating BGN Protein Ubiquitination

doi: 10.1155/2022/5055684

Figure Lengend Snippet: SEMA3B-AS1 was downregulated in human GC tissues. It was mainly located in the nucleus. Data are shown as mean ± SEM ( n = 3); ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. (a) lncRNA expression was identified in three pairs of GC tissues and corresponding PM of GC tissues by microarray analysis according to the value of the samples and fluorescence intensity. (b) The relative mRNA expression of SEMA3B-AS1 in five GC cell lines and normal stomach epithelial cell line (GES-1) was identified by qRT-PCR assay. (c) The relative mRNA expression of SEMA3B-AS1 was identified by qRT-PCR assay in 50 pairs GC tissues and adjacent normal tissues. (d) The intracellular location of SEMA3B-AS1 was identified by RNA FISH assay (magnification: ×400). (e) The nuclear and cytoplasmic distribution of SEMA3B-AS1 in HGC-27 and SGC-7901 cell lines was identified using qRT-PCR assay. (f) The correlation with the expression level of SEMA3B-AS1 between five-year overall survival (OS) and five-year progression-free survival (PFS) was analyzed through Kaplan-Meier survival analysis. (g) The correlation with (PM) between five-year overall survival (OS) and five-year progression-free survival (PFS) was analyzed through Kaplan-Meier survival analysis.

Article Snippet: The construction, synthesis, sequencing, and identification of the plasmid and lentivirus of SEMA3B-AS1 containing green fluorescent protein (GFP) were completed by GenePharma Biotechnology Co. Ltd.

Techniques: Expressing, Microarray, Fluorescence, Quantitative RT-PCR

Journal: Oxidative Medicine and Cellular Longevity

Article Title: The lncRNA SEMA3B-AS1/HMGB1/FBXW7 Axis Mediates the Peritoneal Metastasis of Gastric Cancer by Regulating BGN Protein Ubiquitination

doi: 10.1155/2022/5055684

Figure Lengend Snippet: The correlation of SEMA3B-AS1 expression with the clinicopathological parameters of GC patients ( n = 50).

Article Snippet: The construction, synthesis, sequencing, and identification of the plasmid and lentivirus of SEMA3B-AS1 containing green fluorescent protein (GFP) were completed by GenePharma Biotechnology Co. Ltd.

Techniques: Expressing

Journal: Oxidative Medicine and Cellular Longevity

Article Title: The lncRNA SEMA3B-AS1/HMGB1/FBXW7 Axis Mediates the Peritoneal Metastasis of Gastric Cancer by Regulating BGN Protein Ubiquitination

doi: 10.1155/2022/5055684

Figure Lengend Snippet: Univariate and multivariate analyses of clinicopathologic parameters correlated with progression-free survival (PFS) and overall survival (OS).

Article Snippet: The construction, synthesis, sequencing, and identification of the plasmid and lentivirus of SEMA3B-AS1 containing green fluorescent protein (GFP) were completed by GenePharma Biotechnology Co. Ltd.

Techniques: Expressing

Journal: Oxidative Medicine and Cellular Longevity

Article Title: The lncRNA SEMA3B-AS1/HMGB1/FBXW7 Axis Mediates the Peritoneal Metastasis of Gastric Cancer by Regulating BGN Protein Ubiquitination

doi: 10.1155/2022/5055684

Figure Lengend Snippet: SEMA3B-AS1 could inhibit the activity, invasion, proliferation, and migration and EMT ability of GC cells in vitro (magnification: ×200). Data were shown as mean ± SEM ( n = 3); ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. (a) The overexpression efficiency of Lv-Oe-SEMA3B-AS1 and negative control (NC) was detected in HGC-27 using qRT-PCR. (b) The expression efficiency of Lv-SH-SEMA3B-AS1 and negative control (NC) was detected in SGC-7901 using qRT-PCR. (c) The viability of HGC-27 cells after SEMA3B-AS1 overexpression and negative control (NC) was detected by CCK-8 assay. (d-f) The cell proliferation, migration and invasion ability of GC cells (HGC-27) negative control and SEMA3B-AS1 overexpression were detected by the colony formation assay (d), wound healing assay (e), and transwell assay (f), respectively. (g) The viability of SGC-7901 cells after SEMA3B-AS1 knockdown and negative control (NC) was detected by CCK-8 assay. (h-j) The cell proliferation,migration , and invasion ability of GC cells (SGC-7901) negative control and SEMA3B-AS1 knockdown were detected by the colony formation assay (h), wound healing assay (i), and transwell assay (J), respectively. (k) The expression of EMT markers (E-cadherin and vimentin) in GC cells (HGC-27) was analyzed by Western blotting assay after SEMA3B-AS1 overexpression and negative control. (l) The expression of EMT markers (E-cadherin and vimentin) in GC cells (SGC-7901) was analyzed by Western blotting assay after SEMA3B-AS1 knockdown and negative control.

Article Snippet: The construction, synthesis, sequencing, and identification of the plasmid and lentivirus of SEMA3B-AS1 containing green fluorescent protein (GFP) were completed by GenePharma Biotechnology Co. Ltd.

Techniques: Activity Assay, Migration, In Vitro, Over Expression, Negative Control, Quantitative RT-PCR, Expressing, CCK-8 Assay, Colony Assay, Wound Healing Assay, Transwell Assay, Western Blot

Journal: Oxidative Medicine and Cellular Longevity

Article Title: The lncRNA SEMA3B-AS1/HMGB1/FBXW7 Axis Mediates the Peritoneal Metastasis of Gastric Cancer by Regulating BGN Protein Ubiquitination

doi: 10.1155/2022/5055684

Figure Lengend Snippet: SEMA3B-AS1 can combine with HMGB1. Data are shown as mean ± SEM ( n = 3); ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. (a) RNA pull-down assay showed that biotinylated SEMA3B-AS1 could bind with HMGB1 in GC cells (HGC-27) in vitro. RNA immunoprecipitation (RIP) assay demonstrated the direct binding relationship between SEMA3B-AS1 and HMGB1 in (b) HGC-27 and (c) SGC-7901 cells; the RIP RNA products were detected by qRT-PCR for SEMA3B-AS1. The fold enrichment of SEMA3B-AS1 was relative to its corresponding IgG control. (d) The enrichment of SEMA3B-AS1 in the probe group targeting SEMA3B-AS1 relative to NC probe in HGC-27 cells, as detected by ChIRP assay. GAPDH served as a negative control (NC). (e) The enrichment of HMGB1 protein in the probe group targeting SEMA3B-AS1 relative to the NC probe in HGC-27 cells, as detected by ChIRP assay and the protein product was identified through Western blotting.

Article Snippet: The construction, synthesis, sequencing, and identification of the plasmid and lentivirus of SEMA3B-AS1 containing green fluorescent protein (GFP) were completed by GenePharma Biotechnology Co. Ltd.

Techniques: Pull Down Assay, In Vitro, Immunoprecipitation, Binding Assay, Quantitative RT-PCR, Negative Control, Western Blot

Journal: Oxidative Medicine and Cellular Longevity

Article Title: The lncRNA SEMA3B-AS1/HMGB1/FBXW7 Axis Mediates the Peritoneal Metastasis of Gastric Cancer by Regulating BGN Protein Ubiquitination

doi: 10.1155/2022/5055684

Figure Lengend Snippet: SEMA3B-AS1 promotes the expression of FBXW7 by binding HMGB1. Data are shown as mean ± SEM ( n = 3); ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. (a) The efficiency of HMGB1 knockdown was verified in SGC cells through qRT-PCR assay. (b) The efficiency of HMGB1 overexpression was verified in HGC cells through qRT-PCR assay. The expression levels of HMGB1 protein in the nucleus and cytoplasm of SEMA3B-AS1 (c) overexpressed or (d) interfered cells were detected by Western blotting. (e) The localization of HMGB1 was analyzed by immunofluorescence when SEMA3B-AS1 was overexpressed. Red fluorescence represents HMGB1. (f) The localization of HMGB1 was analyzed by immunofluorescence when SEMA3B-AS1 was knocked down. Red fluorescence represents HMGB1. (g) Dual-Luciferase Assays to assess FBXW7 promoter activity while SEMA3B-AS1 was either alone or in combination with HMGB1. (h) The changes of FBXW7 protein were detected by Western blotting after overexpression or interference of HMGB1 in SEMA3B-AS1-overexpressed gastric cancer cells.

Article Snippet: The construction, synthesis, sequencing, and identification of the plasmid and lentivirus of SEMA3B-AS1 containing green fluorescent protein (GFP) were completed by GenePharma Biotechnology Co. Ltd.

Techniques: Expressing, Binding Assay, Quantitative RT-PCR, Over Expression, Western Blot, Immunofluorescence, Fluorescence, Luciferase, Activity Assay

Journal: Oxidative Medicine and Cellular Longevity

Article Title: The lncRNA SEMA3B-AS1/HMGB1/FBXW7 Axis Mediates the Peritoneal Metastasis of Gastric Cancer by Regulating BGN Protein Ubiquitination

doi: 10.1155/2022/5055684

Figure Lengend Snippet: SEMA3B-AS1 might destabilize the BGN protein by regulating FBXW7, thus promoting the ubiquitination degradation of BGN and inhibiting GC. Data were shown as mean ± SEM ( n = 3); ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. (a) The expression of BGN protein in gastric cancer and adjacent normal tissues was analyzed by bioinformatics using GSE54129 data set. (b) The relative mRNA expression of BGN was identified by qRT-PCR assay in 50 pairs of GC tissues and adjacent normal tissues. (c) The representative IHC staining image for BGN in the paracancerous normal tissues, GC tissues, and GS PM tissues. (d) The relative mRNA expression of FBXW7 in five GC cell lines and normal stomach epithelial cell line (GES-1) was detected by qRT-PCR assay. (e) The overexpression efficiency of FBXW7 and negative control (NC) was detected in HGC-27 using qRT-PCR. (f) The knockdown efficiency of FBXW7 and negative control (NC) was detected in SGC-7901 using qRT-PCR. The mRNA expression levels of BGN were detected by qRT-PCR assay after FBXW7 (g) overexpression or (h) knockdown in GC cells (HGC-27 or SGC-7901). The protein expression levels of BGN were detected by Western blotting assay after FBXW7 (i) overexpression or (j) knockdown in GC cells (HGC-27 or SGC-7901). (k) The interaction between FBXW7 and BGN was identified in HGC-27 cells by coimmunoprecipitation (co-IP). SDS-PAGE separated the immunoprecipitates of input (20%) and BGN. Western blotting was performed to confirm the interaction between FBXW7 and BGN. (l) Cell groups were treated with the proteasome inhibitor MG-132 (50 μ M) or the protein synthesis inhibitor cycloheximide CHX (100 μ g/mL) at different time points (1 h, 4 h, 8 h, and 12 h), and the changes of BGN protein at different time points were detected by Western blotting assay. (m) HGC-27 or SGC-7901 cells were treated with MG-132 (50 μ M) and transfected with HA-Ubi and simultaneously overexpression or knockdown FBXW7 as well as SEMA3B-AS1 overexpressed or knocked down, respectively, FBXW7 vector served as a negative control. Cell lysates were immunoprecipitated with anti-BGN antibody to identify ubiquitination of BGN with anti-HA antibodies using Western blotting assay. (n) The mRNA relative expression levels of SEMA3B-AS1 and BGN were obvious negative association in GC patients using Pearson's correlation analysis (Rs = −0.5568, p < 0.0001).

Article Snippet: The construction, synthesis, sequencing, and identification of the plasmid and lentivirus of SEMA3B-AS1 containing green fluorescent protein (GFP) were completed by GenePharma Biotechnology Co. Ltd.

Techniques: Expressing, Quantitative RT-PCR, Immunohistochemistry, Over Expression, Negative Control, Western Blot, Co-Immunoprecipitation Assay, SDS Page, Transfection, Plasmid Preparation, Immunoprecipitation

Journal: Oxidative Medicine and Cellular Longevity

Article Title: The lncRNA SEMA3B-AS1/HMGB1/FBXW7 Axis Mediates the Peritoneal Metastasis of Gastric Cancer by Regulating BGN Protein Ubiquitination

doi: 10.1155/2022/5055684

Figure Lengend Snippet: SEMA3B-AS1 could inhibit GC tumorigenesis and PM in vivo. Representative images of migratory or invaded cells (magnification, ×200) were shown. Data are shown as mean ± SEM ( n = 3); ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. The morphological properties of (a) tumor subcutaneous xenograft, (b) tumor size, and (c) tumor weight in OeNC-SEMA3B-AS1-HGC-27, Oe-SEMA3B-AS1, and Oe-SEMA3B-AS1+SH-FBXW7-HGC-27 cells at 35 days, each group had five mice. (d) IHC analyzed the expression of FBXW7 and BGN protein of tumors from the OeNC-SEMA3B-AS1-HGC-27, Oe-SEMA3B-AS1, and Oe-SEMA3B-AS1+SH-FBXW7-HGC-27 cell groups. (e) The representative image of intraperitoneal tumor formation model from OeNC-SEMA3B-AS1-HGC-27 and Oe-SEMA3B-AS1-HGC-27. (f) Simultaneously, we counted and analyzed the number of peritoneal, perigastric, intestinal and mesenteric, and diaphragmatic metastases, each group had six mice. (g) The proposed mechanism model in which the SEMA3B-AS1/HMGB1/FBXW7 axis mediates the PM of GC by regulating BGN protein ubiquitination.

Article Snippet: The construction, synthesis, sequencing, and identification of the plasmid and lentivirus of SEMA3B-AS1 containing green fluorescent protein (GFP) were completed by GenePharma Biotechnology Co. Ltd.

Techniques: In Vivo, Expressing

Journal: Communications Biology

Article Title: Aging-regulated PNUTS maintains endothelial barrier function via SEMA3B suppression

doi: 10.1038/s42003-024-06230-5

Figure Lengend Snippet: Primers used in this study

Article Snippet: In order to quantify SEMA3B levels, 300 µl supernatant was applied to a human SEMA3B ELISA plate (DLdevelop, Shuigoutou, China), the SEMA3B protein standard was diluted in full medium.

Techniques: Mutagenesis

Journal: Communications Biology

Article Title: Aging-regulated PNUTS maintains endothelial barrier function via SEMA3B suppression

doi: 10.1038/s42003-024-06230-5

Figure Lengend Snippet: a The two subsets of RNAseq data we obtained, from endothelial-depleted PNUTS mouse lungs and PNUTS knockdown (KD) HUVECs, were compared to find common targets of PNUTS depletion. The Venn diagram depicts the finding of 277 common transcripts, which were functionally analysed using KEGG pathways analysis, shown in the table below. b Changes in axon guidance gene expression after PNUTS silencing was assessed by RT-qPCR. Expression values are relative siControl-treated HUVECs and normalized to RPSA mRNA ( n = 6). c Volcano plot showing the distribution of gene expression in PNUTS KD versus control HUVECs. SEMA3B is marked in red. d Supernatant SEMA3B concentration was determined by ELISA 72 h after PNUTS silencing ( n = 4). e Expression levels of Sema3b mRNA in intima samples of PNUTS EC-KO mice was assessed by RT-qPCR, relative to WT samples and normalized to Rplp0 mRNA. f HUVECs were treated with vehicle or Tautomycetin (166 nM) for 48 h and mRNA was analyzed by RT-qPCR for expression of SEMA3B, normalized to RPSA mRNA ( n = 3). g Expression of SEMA3B was measured in mRNA samples of HUVECs assayed in Fig. , relative to control cells and normalized to RPSA mRNA. h - i HUVECs were co-transfected with siPNUTS and/or siSEMA3B were subjected to ECIS for 48 h ( n = 4 independent experiments, 4 biological replicates per group and experiment). h Cell-cell interaction was modeled. i Endothelial resistance was measured at 4000 Hz. j Top panel: siSEMA3B rescues the effect of PNUTS silencing on adherence junctions (shown as PECAM1 IF staining). Bottom panel: PECAM1 IF staining shows time course of change in adherens junctions upon stimulation with recombinant human SEMA3B. k Cell-cell interaction was modeled using ECIS. The arrow indicates the 48 h time point at which recombinant SEMA3B was added to the medium (or not; untreated). Quantification was performed at 60 h ( n = 4 biological replicates and 3–4 technical replicates). l Graphic summary of the proposed mechanism. In young individuals, PNUTS interacts and promotes activity of PP1, which represses the expression of SEMA3B. Endothelial cells are in homeostasis and maintain their barrier function. During aging, PNUTS is repressed in endothelial cells. The absence of PNUTS inhibits PP1 function at the SEMA3B promoter activating SEMA3B expression. SEMA3B exerts repulsive signals between endothelial cells, promoting intercellular gaps and disrupting the barrier. This provokes a series of critical changes in the cells leading to cellular senescence. * p < 0.05, ** p < 0.01, *** p < 0.001. Error bars depict the standard error of the mean (SEM).

Article Snippet: In order to quantify SEMA3B levels, 300 µl supernatant was applied to a human SEMA3B ELISA plate (DLdevelop, Shuigoutou, China), the SEMA3B protein standard was diluted in full medium.

Techniques: Expressing, Quantitative RT-PCR, Concentration Assay, Enzyme-linked Immunosorbent Assay, Transfection, Staining, Recombinant, Activity Assay