Journal: Disease Markers

Article Title: The Key Genes Underlying Pathophysiology Association between Plaque Instability and Progression of Myocardial Infarction

doi: 10.1155/2021/4300406

Figure Lengend Snippet: Validations of screened hub genes. The protein expression levels of left ventricle at 1 d, 1 w, and 8 w after MI utilizing ELISA, including PAK3 (a), RASD2 (b), IQGAP2 (c), SLIT2 (d), CD44 (e), CD5 (f), SOCS3 (g), PIK3CD (h), VAV1 (i), and P2RY1 (j).

Article Snippet: For further validation, the left ventricular sample levels, including IQGAP2, Pak3, Slit2, CD44, CD5, SOCS3, and P2RY1, were measured using a mouse IQGAP2 ELISA kit (ybE288Mu, Ambion, China), mouse Pak3 ELISA kit (CSB-PA017407LA01HU, CUSABIO), mouse Slit2 ELISA kit (Q9R1B9, RayBiotech), mouse CD44 ELISA kit (P15379, RayBiotech), mouse CD5 ELISA kit (Q91X69, RayBiotech), mouse SOCS3 ELISA kit (EKU07517, BIOMATIK), mouse Vav1 ELISA kit (JN-S-86921, AFZHAN), and mouse P2RY1 ELISA kit (CSB-EL017326MO, CUSABIO), respectively.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay

Journal: Oncotarget

Article Title: Slit2/Robo1 signaling promotes intestinal tumorigenesis through Src-mediated activation of the Wnt/β-catenin pathway

doi:

Figure Lengend Snippet: Following methylene blue staining, the small intestines from the Apc Min/+ and Apc Min/+ ; Slit2 mice were examined macroscopically (A). Overexpression of Slit2 significantly increased the number (B) and size (C) of the tumors. Overexpression of Slit2 resulted in a marked increase in cell proliferation as shown by enhanced BrdU uptake in the tumor tissues of Apc Min/+ ; Slit2 mice (D). The results of IHC were better presented quantitatively using IPP software (D, right panel). On the other hand, inactivation of the Slit2/Robo1 signaling by siRNA-mediated gene knockdown led to a reduced proliferation of SW620 and SW480 cells (n=6 per group); conversely, overexpression of Slit2 or Robo1 caused an increased proliferation of HCT-116 cells (E). The results of methylene blue staining and IHC staining are representative of 11 mice per group (All mice were 24-week-old). The statistical data were expressed as the mean ±S.D. **: P < 0.01, and ***: P < 0.001. Scale bars: 20 μm.

Article Snippet: The siRNA duplex for Slit2 was obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, California, USA), and the siRNA duplexes for Robo1, Src and E-cadherin were obtained from Guangzhou RiboBio Co., Ltd. (Guangzhou, China).

Techniques: Staining, Over Expression, Software, Immunohistochemistry

Journal: Oncotarget

Article Title: Slit2/Robo1 signaling promotes intestinal tumorigenesis through Src-mediated activation of the Wnt/β-catenin pathway

doi:

Figure Lengend Snippet: Expression of β-catenin, CCND1 and Axin2 in the tumor tissues of Apc Min/+ and Apc Min/+ ; Slit2 mice was examined by IHC, and the results were quantitatively determined using IPP software and expressed as the mean ± S.D. (A). The subcellular location of β-catenin was examined in the tumor tissues of Apc Min/+ and Apc Min/+ ; Slit2 mice by IF (B). Inactivation of Slit2/Robo1 signaling by suppressing Slit2 or Robo1 expression inhibits the expression of β-catenin in SW620 and SW480 cells, but conversely, activation of Slit2/Robo1 signaling through overexpressing Slit2 or Robo1 expression promotes β-catenin expression in HCT-116 cells (C). Inactivation of Slit2/Robo1 signaling by siRNA against Slit2 or Robo1 suppresses nuclear translocation of β-catenin in SW620 cells, and overexpression of Slit2 or Robo1 promotes nuclear translocation of β-catenin in HCT-116 cells (D). All results represent at least three separate experiments (C and D). The IHC and IF staining of the mouse tissues are representative of 11 mice per group (All mice were 24-week-old). The results of IHC were determined using IPP software, and expressed as the mean ± S.D. *: P < 0.05, ***: P < 0.001. Scale bars, 20 μm (A) and 25 μm (B and D).

Article Snippet: The siRNA duplex for Slit2 was obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, California, USA), and the siRNA duplexes for Robo1, Src and E-cadherin were obtained from Guangzhou RiboBio Co., Ltd. (Guangzhou, China).

Techniques: Expressing, Software, Activation Assay, Translocation Assay, Over Expression, Staining

Journal: Oncotarget

Article Title: Slit2/Robo1 signaling promotes intestinal tumorigenesis through Src-mediated activation of the Wnt/β-catenin pathway

doi:

Figure Lengend Snippet: IHC analysis of the expression of total Src and pSrc (Tyr416) in the tumor tissues of Apc Min/+ and Apc Min/+ ; Slit2 mice (A). Inactivation of Slit2/Robo1 significantly reduced the expression of pSrc (Tyr 416) in SW620 and SW480 cells, and activation of Slit2/Robo1 signaling through overexpressing Slit2 or Robo1 expression promotes pSrc (Tyr 416) expression in HCT-116 cells (B). IHC analysis of the expression of E-cadherin in the tumor tissues of the Apc Min/+ and Apc Min/+ ; Slit2 mice (C). Inactivation of Src signaling significantly enhances the expression of E-cadherin but inhibits the expression of β-cateninin in SW620 and SW480 cells (D). Src inactivation in SW620 and SW480 cells also led to a reduced nuclear translocation of β-catenin (E). Inhibition of E-cadherin expression through siRNA technique could promote the expression of β-cateninin and vimentin in SW620 and SW480 cells (F). IHC analysis of the expression of vimentin in the tumor tissues of Apc Min/+ and Apc Min/+ ; Slit2 mice (G). The data in IHC staining are representative of 11 mice per group (All mice were 24-week-old). The results of IHC were determined using IPP software, and the data were expressed as the mean ±S.D. *: P < 0.05, **: P < 0.01. Scale bars: 20 μm (A, C and G) and 25 μm (E).

Article Snippet: The siRNA duplex for Slit2 was obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, California, USA), and the siRNA duplexes for Robo1, Src and E-cadherin were obtained from Guangzhou RiboBio Co., Ltd. (Guangzhou, China).

Techniques: Expressing, Activation Assay, Translocation Assay, Inhibition, Immunohistochemistry, Software

Journal: Neural Development

Article Title: The microtubule plus-end-tracking protein TACC3 promotes persistent axon outgrowth and mediates responses to axon guidance signals during development

doi: 10.1186/s13064-017-0080-7

Figure Lengend Snippet: TACC3 affects axon guidance in vivo and ex vivo. a , b , confocal images of laterally-viewed whole-mount Xenopus spinal cord fluorescently labeled for acetylated tubulin, showing peripheral axon outgrowth in control ( a ) and TACC3 KD ( b ) embryos at 2 dpf. c , Quantitation of the embryos with motor neuron guidance defects ( n = 5 embryos). d , Representative neural tube growth cone images of control and TACC3 OE, before and after addition of 400 ng/ml Slit2. e , Quantification of the percentage of the growth cone collapse events in control ( n = 48) and TACC3 OE ( n = 82) growth cones show significant reduction in growth cone collapse in TACC3 overexpressing growth cones. f, Cartoon model for the role of TACC3 at MT plus ends during axon outgrowth and guidance. Microtubule ( blue ) plus-ends decorated by TACC3 ( green ) promotes axon outgrowth, reduces axon retraction, dampens nocodazole induced reduction in MT dynamics parameters, rescues XMAP215 KD induced axon length reduction and opposes repellent guidance signals effect. * p < 0.05, ** p < 0.01, *** p < 0.001. ns not significant. n = growth cone number. Scale bar, 50 μm and 5 μm

Article Snippet: Recombinant mouse Slit2 protein (R&D Systems) (400 ng/ml) was administered to cultured neural tube explants derived from stage 28 Xenopus embryos in 400 μl culture media supplemented with 1% Penicillin/Streptomycin and 0.1% BSA.

Techniques: In Vivo, Ex Vivo, Labeling, Quantitation Assay

Journal: eLife

Article Title: The chemorepellent, SLIT2, bolsters innate immunity against Staphylococcus aureus

doi: 10.7554/eLife.87392

Figure Lengend Snippet:

Article Snippet: Human SLIT2 ELISA (for HMEC-1 conditioned media) (#CSB-E11038h, CUSABIO, Wuhan, China) was performed according to the manufacturer’s instructions using a validated kit ( ).

Techniques: Western Blot, Blocking Assay, Flow Cytometry, Control, In Vivo, Injection, Sequencing, Sterility, Bacteria, Electron Microscopy, Isolation, SYBR Green Assay, Real-time Polymerase Chain Reaction, Activation Assay, Recombinant, Reverse Transcription, Staining, Polymer, Enzyme-linked Immunosorbent Assay, Software

Journal: Frontiers in Cell and Developmental Biology

Article Title: Obesity Inhibits Angiogenesis Through TWIST1-SLIT2 Signaling

doi: 10.3389/fcell.2021.693410

Figure Lengend Snippet: TWIST1 expression in obese human subcutaneous adipose ECs is lower than lean ECs. (A) Volcano plot of differentially expressed genes, including TWIST1, in eight obese vs. seven lean adult human subcutaneous adipose samples from the GSE55200 dataset. Blue indicates significantly down-regulated genes and red indicates significantly up-regulated genes with Benjamini and Hochberg adjusted p -values < 0.05. (B) Graph showing the TWIST1 mRNA levels in ECs isolated from lean (BMI < 30) vs. obese (BMI > 30) human adipose tissues ( n = 9–10, mean ± SEM, * p < 0.05). The numbers along the dots correspond to the clone numbers in . (C) Immunoblots showing the TWIST1 and β-Actin protein levels in ECs isolated from lean (BMI < 30) vs. obese (BMI > 30) human adipose tissues ( n = 5, mean ± SEM, * p < 0.05). The numbers along the dots correspond to the clone numbers in . (D) Network showing connections among TWIST1, SLIT2, and genes from the top 30 BP GO Term categories derived from 1,302 significantly differentially expressed down and upregulated genes with Benjamini and Hochberg adjusted p -values < 0.05 from the GSE55200 dataset. Genes are color-coded by GO Term categories. Red, angiogenesis-related genes; Orange, development-related genes; Gold, ECM and cell adhesion-related genes; Dark green, cell proliferation, migration-related genes; Blue, inflammation and/or immune response-related genes; Purple, cell signaling and/or apoptosis-related genes; Gray, miscellaneous category-related genes; Black outlines, genes from the GSE55200 dataset.

Article Snippet: Recombinant human SLIT2 (aa1122–1529) was purchased from R&D Systems (Minneapolis, MN, United States).

Techniques: Expressing, Isolation, Western Blot, Derivative Assay, Migration

Journal: Frontiers in Cell and Developmental Biology

Article Title: Obesity Inhibits Angiogenesis Through TWIST1-SLIT2 Signaling

doi: 10.3389/fcell.2021.693410

Figure Lengend Snippet: TWIST1 controls SLIT2 expression in lean vs. obese adipose ECs. (A) Graph showing SLIT2 mRNA expression in lean vs. obese human adipose ECs ( n = 9–10, mean ± SEM, * p < 0.05). (B) Graph showing SLIT2 protein expression in lean vs. obese human adipose tissues ( n = 9, mean ± SEM, * p < 0.05). (C) Graphs showing mRNA expression of TWIST1 and SLIT2 in lean human adipose ECs treated with TWIST1 siRNA or control siRNA with irrelevant sequences ( n = 4–5, mean ± SEM, * p < 0.05). Graph showing protein expression of SLIT2 in lean human adipose ECs treated with TWIST1 siRNA or control siRNA with irrelevant sequences ( n = 4, mean ± SEM, * p < 0.05). (D) Graphs showing mRNA expression of TWIST1 and SLIT2 in obese human adipose ECs treated with lentivirus overexpressing TWIST1 or control virus ( n = 4, mean ± SEM, * p < 0.05). Graph showing protein expression of SLIT2 in obese human adipose ECs treated with lentivirus overexpressing TWIST1 or control virus ( n = 4, mean ± SEM, * p < 0.05). The numbers along the dots correspond to the clone numbers in .

Article Snippet: Recombinant human SLIT2 (aa1122–1529) was purchased from R&D Systems (Minneapolis, MN, United States).

Techniques: Expressing, Virus

Journal: Frontiers in Cell and Developmental Biology

Article Title: Obesity Inhibits Angiogenesis Through TWIST1-SLIT2 Signaling

doi: 10.3389/fcell.2021.693410

Figure Lengend Snippet: TWIST1-SLIT2 signaling controls DNA synthesis and EC migration in lean vs. obese human adipose ECs. (A) Representative images showing EdU positive (green) lean (BMI < 30) vs. obese (BMI > 30) human adipose ECs ( left ). Scale bar, 50 μm. Graph showing DNA synthesis of lean (BMI < 30) vs. obese (BMI > 30) human adipose ECs analyzed using an EdU staining assay ( right , n = 6, mean ± SEM, * p < 0.05). (B) Representative images showing lean (BMI < 30) vs. obese (BMI > 30) human adipose ECs migrated to the opposite side of the trans -well membrane and stained with Wright Giemsa solution ( left ). Scale bar, 50 μm. Graph showing lean vs. obese human adipose ECs migrating toward 5% FBS ( right , n = 5, mean ± SEM, * p < 0.05). (C) Representative images showing EdU positive (green) lean (BMI < 30) human adipose ECs treated with TWIST1 siRNA ( left ). Scale bar, 50 μm. Graph showing EdU-positive lean (BMI < 30) human adipose ECs (clone #6) treated with TWIST1 siRNA ( right , n = 3, mean ± SEM, * p < 0.05). As a control, human adipose ECs were treated with siRNA with irrelevant sequences. (D) Representative images showing lean (BMI < 30) human adipose ECs treated with TWIST1 siRNA, migrated to the opposite side of the trans-well membrane and stained with Wright Giemsa solution ( left ). Scale bar, 50 μm. Graph showing lean human adipose ECs (clone #6) treated with TWIST1 siRNA migrating toward 5% FBS ( right , n = 5, mean ± SEM, * p < 0.05). As a control, human adipose ECs were treated with siRNA with irrelevant sequences. (E) Graph showing EdU-positive obese (BMI > 30) human adipose ECs treated with lentivirus overexpressing TWIST1 or in combination with SLIT2 shRNA ( n = 6–9, mean ± SEM, * p < 0.05). As a control, human adipose ECs were treated with control virus (vector alone). (F) Representative images showing EdU positive obese (BMI > 30) human adipose ECs treated with lentivirus overexpressing TWIST1 or in combination with SLIT2 shRNA. Scale bar, 50 μm. (G) Graph showing obese human adipose ECs treated with lentivirus overexpressing TWIST1 or in combination with SLIT2 shRNA migrating toward 5% FBS ( n = 5–7, mean ± SEM, * p < 0.05). As a control, human adipose ECs were treated with control virus (vector alone). (H) Representative images showing obese (BMI > 30) human adipose ECs treated with lentivirus overexpressing TWIST1 or in combination with SLIT2 shRNA, migrated to the opposite side of the trans -well membrane and stained with Wright Giemsa solution. Scale bar, 50 μm. (I) Graph showing SLIT2 mRNA levels in obese human adipose ECs treated with lentivirus expressing SLIT2 shRNA ( n = 6, mean ± SEM, * p < 0.05). As a control, human adipose ECs were treated with control virus (vector alone). The numbers along the dots correspond to the clone numbers in .

Article Snippet: Recombinant human SLIT2 (aa1122–1529) was purchased from R&D Systems (Minneapolis, MN, United States).

Techniques: DNA Synthesis, Migration, Staining, Membrane, shRNA, Virus, Plasmid Preparation, Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Obesity Inhibits Angiogenesis Through TWIST1-SLIT2 Signaling

doi: 10.3389/fcell.2021.693410

Figure Lengend Snippet: TWIST1-SLIT2 signaling mediates obesity-dependent inhibition of vascular formation in the gel subcutaneously implanted on mice. (A) 3D reconstructed immunofluorescence (IF) images showing GFP-labeled vascular formation and DAPI in the fibrin gel supplemented with GFP-labeled lean (BMI < 30, clone #15) vs. obese (BMI > 30, clone #7) human adipose ECs and subcutaneously implanted on NSG mice for 7 days. Scale bar, 50 μm. Graphs showing vascular area and average vessel length in the gel ( n = 5–6, mean ± SEM, * p < 0.05). (B) 3D reconstructed IF micrographs showing GFP-labeled vascular formation and DAPI in the fibrin gel supplemented with GFP-labeled lean human adipose ECs (clone #15) treated with lentivirus encoding SLIT2 shRNA and subcutaneously implanted on NSG mice for 7 days. As a control, lean human adipose ECs were treated with control virus. Scale bar, 50 μm. Graphs showing vascular area and average vessel length in the gel ( n = 6–7, mean ± SEM, * p < 0.05). (C) 3D reconstructed IF micrographs showing GFP-labeled vascular formation and DAPI in the fibrin gel supplemented with GFP-labeled obese human adipose ECs (clone #7) treated with SLIT2 protein, lentivirus overexpressing TWIST1 or in combination with SLIT2 shRNA and subcutaneously implanted on NSG mice for 7 days. As a control, obese human adipose ECs were treated with control virus (vector alone), control shRNA, or control vehicle. Scale bar, 50 μm. Graphs showing vascular area and average vessel length in the gel ( n = 6–7, mean ± SEM, * p < 0.05).

Article Snippet: Recombinant human SLIT2 (aa1122–1529) was purchased from R&D Systems (Minneapolis, MN, United States).

Techniques: Inhibition, Immunofluorescence, Labeling, shRNA, Virus, Plasmid Preparation