antibodies against sema3b  (Novus Biologicals)

Journal: MedComm

Article Title: SEMA3B‐AS1 suppresses colorectal carcinoma progression by inhibiting Semaphorin 3B‐dependent VEGF signaling pathway activation

doi: 10.1002/mco2.365

Figure Lengend Snippet: Long noncoding RNA ( LncRNA) SEMA3B‐AS1 could be an important prognostic factor in predicting the survival of colorectal carcinoma (CRC) patients . (A) Hierarchical clustering analysis of differentially expressed lncRNAs that intersected between CRC tissues and paracancerous normal intestinal mucosal tissues, as well as between CRC cells SW620 and SW480. N, normal intestinal mucosa tissues. C, colorectal carcinoma tissues. The red square highlights SEMA3B‐AS1 . (B) The level of SEMA3B‐AS1 in paired colorectal carcinoma and adjacent noncancerous tissues. (C) The level of SEMA3B‐AS1 in colorectal carcinoma and colon mucosa epithelial (FHC) cell lines. (D) Expression analysis of SEMA3B‐AS1 in normal colorectal mucosa and colorectal carcinoma tissues by in situ hybridization (ISH). Scale bars are shown in the right–left corner of each picture. The scale bar indicates 100 μm in upper row, and the scale bar indicates 20 μm in lower row. (E) Correlation among TNM, lymph node metastasis, distant metastasis, and expression of SEMA3B‐AS1 . (F and G) Kaplan–Meier survival analysis in all patients with colorectal carcinoma according to SEMA3B‐AS1 expression according to our data (F) and The Cancer Genome Atlas (TCGA) cohort (G). For (B)–(E), data are presented as means ± SD in three independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The slides were incubated at 4°C for 8 h with primary antibodies against SEMA3B (1:100, NB100‐2218SS, Novusbio) and Ki‐67 (1:500, 27309‐1‐AP, ProteinTech Group).

Techniques: Expressing, In Situ Hybridization

Journal: MedComm

Article Title: SEMA3B‐AS1 suppresses colorectal carcinoma progression by inhibiting Semaphorin 3B‐dependent VEGF signaling pathway activation

doi: 10.1002/mco2.365

Figure Lengend Snippet: SEMA3B‐AS1 overexpression inhibits colorectal carcinoma cell growth and metastasis in vitro and in vivo. (A) SEMA3B‐AS1 overexpression suppressed cell proliferation in colorectal carcinoma cell lines as determined by CCK‐8 assay. (B) SEMA3B‐AS1 overexpression inhibited colony formation in colorectal carcinoma cells. Representative images (left) and quantitative analyses (right) are shown. (C) SEMA3B‐AS1 overexpression induced cell cycle arrest in the G1 phase in colorectal carcinoma cells. (D) SEMA3B‐AS1 overexpression inhibited colorectal carcinoma cell invasion in a Matrigel invasion assay. Scale bars indicate 50 μm and are shown in the right–left corner of each picture. (E) SEMA3B‐AS1 overexpression suppressed cell migration in the wound‐healing assay. The scale bar indicates 100 μm. The experiments were performed at least three times, and the data are expressed as the mean ± SD. (F) SEMA3B‐AS1 overexpression inhibited subcutaneous tumor formation in nude mice. HCT116 cells with ectopic overexpression of SEMA3B‐AS1 and control cells were inoculated into nude mice ( n = 7 per group). The effect of SEMA3B‐AS1 on colorectal carcinoma tumor growth was evaluated based on tumor volume in the two groups. (G) Representative photographs of hematoxylin and eosin (H&E) and immunohistochemistry (IHC) staining for Ki‐67 in primary cancer tissues. The scale bar indicates 20 μm. (H and I) Intrasplenic injections to establish a liver metastasis model in nude mice ( n = 6 per group). The tumors in spleen and liver metastases after colorectal carcinoma (CRC) cell intrasplenic injections for 4 weeks (H, left) and the statistical distribution of metastasis numbers (H, right) are shown. The tissues were stained by H&E staining (I). The scale bar indicates 100 μm in upper row, and the scale bar indicates 20 μm in lower row. For (A)–(H), data are expressed as the means ± SD of three independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The slides were incubated at 4°C for 8 h with primary antibodies against SEMA3B (1:100, NB100‐2218SS, Novusbio) and Ki‐67 (1:500, 27309‐1‐AP, ProteinTech Group).

Techniques: Over Expression, In Vitro, In Vivo, CCK-8 Assay, Invasion Assay, Migration, Wound Healing Assay, Control, Immunohistochemistry, Staining

Journal: MedComm

Article Title: SEMA3B‐AS1 suppresses colorectal carcinoma progression by inhibiting Semaphorin 3B‐dependent VEGF signaling pathway activation

doi: 10.1002/mco2.365

Figure Lengend Snippet: SEMA3B, the sense‐cognate gene for SEMA3B‐AS1 , is a key downstream target of SEMA3B‐AS1 . (A) Genomic location of SEMA3B and SEMA3B‐AS1 from the ENCODE collection. Higher levels of epigenetic modification marks on histone 3 at lysine 9 (H3K9ac) and several transcription factor binding site uniform peaks of EP300 were observed within the SEMA3B promoter region. (B and C) The correlation between SEMA3B‐AS1 transcript levels and SEMA3B mRNA levels in colorectal carcinoma tissues was measured according to our data ( n = 30; B) and The Cancer Genome Atlas (TCGA) cohort ( n = 622; C). (D and E) SEMA3B‐AS1 regulated the expression of SEMA3B at the mRNA (D) and protein (E) levels. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The slides were incubated at 4°C for 8 h with primary antibodies against SEMA3B (1:100, NB100‐2218SS, Novusbio) and Ki‐67 (1:500, 27309‐1‐AP, ProteinTech Group).

Techniques: Modification, Binding Assay, Expressing

Journal: MedComm

Article Title: SEMA3B‐AS1 suppresses colorectal carcinoma progression by inhibiting Semaphorin 3B‐dependent VEGF signaling pathway activation

doi: 10.1002/mco2.365

Figure Lengend Snippet: SEMA3B‐AS1 promotes the expression of SEMA3B by recruiting EP300 to accelerate histone H3 acetylation. (A) Nuclear‐enriched SEMA3B‐AS1 was determined by fluorescence in situ hybridization (left) and RNA fraction assays (right). (B) RIP experiments were performed in HCT116 cells using an EP300 or nonspecific IgG antibody to determine the amount of SEMA3B‐AS1 RNA associated with EP300 or IgG relative to the input control. (C) RNA pull‐down assays were used to identify proteins associated with SEMA3B‐AS1 . Biotinylated SEMA3B‐AS1 and antisense RNA were incubated with cell extracts, and the associated proteins were resolved by SDS–PAGE. Western blotting was performed to analyze the specific interaction between EP300 and SEMA3B‐AS1 . Dot blot of RNA–protein binding samples indicates equal RNA transcripts present in the assay. (D) SEMA3B‐AS1 did not affect EP300 mRNA expression levels (left), but SEMA3B‐AS1 overexpression significantly increased the protein levels of EP300, H3K9ac, and SEMA3B (right). (E and F) EP300 can bind to the SEMA3B promoter. A schematic illustration of the SEMA3B‐AS1 and SEMA3B structures is shown (E, top). Arrows show the direction of transcription. The numbered sites denote the promoter fragments of the SEMA3B gene. The ability of EP300 to bind to SEMA3B promoter regions was assessed by chromatin immunoprecipitation (ChIP) (E) and was increased as a result of SEMA3B‐AS1 overexpression (F). White arrow shows the SEMA3B promoter region that EP300 binds to (E). (G) Depletion of EP300 mediated by siRNA significantly decreased H3K9 acetylation and SEMA3B protein levels in colorectal carcinoma cell lines overexpressing SEMA3B‐AS1 . (H) SEMA3B mRNA was regulated by the histone deacetylase inhibitor TSA and acetyltransferase inhibitor C646 in colorectal carcinoma cells. The C646 and TSA double‐negative group was set as control. (I) SEMA3B protein was regulated by the histone deacetylase inhibitor TSA and acetyltransferase inhibitor C646 in colorectal carcinoma cells. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The slides were incubated at 4°C for 8 h with primary antibodies against SEMA3B (1:100, NB100‐2218SS, Novusbio) and Ki‐67 (1:500, 27309‐1‐AP, ProteinTech Group).

Techniques: Expressing, Fluorescence, In Situ Hybridization, Control, Incubation, SDS Page, Western Blot, Dot Blot, Protein Binding, Over Expression, Chromatin Immunoprecipitation, Histone Deacetylase Assay

Journal: MedComm

Article Title: SEMA3B‐AS1 suppresses colorectal carcinoma progression by inhibiting Semaphorin 3B‐dependent VEGF signaling pathway activation

doi: 10.1002/mco2.365

Figure Lengend Snippet: SEMA3B‐AS1 requires EP300/SEMA3B to suppress colorectal carcinoma cell growth, invasion, and migration. SEMA3B‐AS1 overexpression significantly reduced the colorectal carcinoma cell proliferation (A), cell cycle progression (B), colony formation (C), invasion (D), and migration (E). The potential effects of SEMA3B‐AS1 on colorectal carcinoma cells were completely abolished, similar to the control cells, by EP300 knockdown by siRNA. The depletion of SEMA3B impairs colorectal carcinoma tumor cell proliferation efficiency and diminishes the suppressive effects of SEMA3B‐AS1 overexpression in a similar manner. Scale bars indicate 50 μm (D) and 100 μm (E). * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The slides were incubated at 4°C for 8 h with primary antibodies against SEMA3B (1:100, NB100‐2218SS, Novusbio) and Ki‐67 (1:500, 27309‐1‐AP, ProteinTech Group).

Techniques: Migration, Over Expression, Control, Knockdown

Journal: MedComm

Article Title: SEMA3B‐AS1 suppresses colorectal carcinoma progression by inhibiting Semaphorin 3B‐dependent VEGF signaling pathway activation

doi: 10.1002/mco2.365

Figure Lengend Snippet: SEMA3B overexpression inhibits angiogenesis and the vascular endothelial growth factor (VEGF) pathway. (A) Gene set enrichment analysis (GSEA) showed the enrichment of the VEGF pathway in CRC cells with SEMA3B downregulation. (B) Overexpression of SEMA3B‐AS1 or SEMA3B increased the content of SEMA3B protein in the supernatant of colorectal cancer cells. (C) Colorectal carcinoma (CRC) cell supernatant overexpressing SEMA3B‐AS1 or SEMA3B inhibited the invasion ability of endothelial cells. Representative images (left) and the statistical analysis (right) are shown. Scale bars indicate 50 μm. (D) CRC cell supernatant overexpressing SEMA3B‐AS1 or SEMA3B inhibited the tube formation of endothelial cells in vitro. Scale bars indicate 100 μm. (E) The chorioallantoic membrane (CAM) assay was used to examine blood vessel formation after stimulation with the supernatants from the indicated cells. The yellow circles indicate locations where conditioned medium was used. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The slides were incubated at 4°C for 8 h with primary antibodies against SEMA3B (1:100, NB100‐2218SS, Novusbio) and Ki‐67 (1:500, 27309‐1‐AP, ProteinTech Group).

Techniques: Over Expression, In Vitro, Membrane, Chick Chorioallantoic Membrane Assay

Journal: MedComm

Article Title: SEMA3B‐AS1 suppresses colorectal carcinoma progression by inhibiting Semaphorin 3B‐dependent VEGF signaling pathway activation

doi: 10.1002/mco2.365

Figure Lengend Snippet: SEMA3B‐AS1 requires SEMA3B/NRP1 to suppress angiogenesis. (A) Protein network analysis of possible SEMA3B interacted proteins. The red square highlights NRP1. (B) SEMA3B and NRP1 colocalize in the blood vessels of colorectal carcinoma (CRC) patients. The scale bars indicate 100 and 20 μm in pictures at 100× or 400× magnifications, respectively. (C) Both SEMA3B and vascular endothelial growth factor (VEGF) interact with NRP1, as analyzed by coIP. (D) NRP1 can bind with more SEMA3B and less VEGF after SEMA3B‐AS1 overexpression. (E–G) SEMA3B‐AS1 required SEMA3B/NRP1 to suppress the human umbilical vein endothelial cell (HUVEC) invasion (E), tubule formation in vitro (F), and angiogenesis in the chorioallantoic membrane (CAM) assay (G). Scale bars indicate 50 μm (E) and 100 μm (F), respectively. The yellow circles in (G) indicate locations where conditioned medium was used. (H) Schematic diagram showing the mechanism of action of SEMA3B‐AS1 in CRC. SEMA3B‐AS1 was downregulated in colorectal carcinoma and consequently decreased EP300 recruitment. In turn, the reduction in EP300 recruitment suppressed the accumulation of the active marker H3K9ac and repressed SEMA3B levels. Subsequently, the receptor NRP1 interacted with less SEMA3B, and then, the VEGF pathway was activated, which induced angiogenesis and colorectal carcinoma progression.* p < 0.05; ** p < 0.01; *** p < 0.001. Source : Illustration created with BioRender (available online: https://biorender.com/ (accessed on 15 June 2022)).

Article Snippet: The slides were incubated at 4°C for 8 h with primary antibodies against SEMA3B (1:100, NB100‐2218SS, Novusbio) and Ki‐67 (1:500, 27309‐1‐AP, ProteinTech Group).

Techniques: Over Expression, In Vitro, Membrane, Chick Chorioallantoic Membrane Assay, Marker