Journal: Molecular omics

Article Title: Serial In-solution digestion protocol for mass spectrometry-based glycomics and proteomics analysis

doi: 10.1039/d0mo00019a

Figure Lengend Snippet: Sample setup, including various standard proteoglycans and proteins, and mouse brain tissue lysate used for testing the protocol for each step i-iv (see ). HSBK; heparan sulfate from bovine kidney, HSPIM; Heparan sulfate from porcine intestinal mucosa, AGP; α-Acid Glycoprotein.

Article Snippet: R&D systems) Mouse brain tissue lysate (89-014-501, Abnova) Human neurocan recombinant protein (6508NC050, R&D systems) Aggrecan from bovine articular cartilage (A1960-1MG, SigmaAldrich) Human Syndecan-1 Protein (#7879, BioVision Incorporated) α1-Acid Glycoprotein from human plasma (#G9885, SigmaAldrich) Pierce retention time calibration mixture (#88321, ThermoScientific) Pierce™ HeLa Protein Digest Standard (#88328, Thermo Scientific) Heparan sulfate from porcine intestinal mucosa (HSPIM) (Celsus Laboratories, Inc.0 Heparan sulfate from bovine kidney (HSBK) (SigmaAldrich)

Techniques:

Journal: Molecular omics

Article Title: Serial In-solution digestion protocol for mass spectrometry-based glycomics and proteomics analysis

doi: 10.1039/d0mo00019a

Figure Lengend Snippet: A, Coverage, total, and unique peptides for standard proteins and proteoglycans as observed by LC-MS/MS analysis after serial in-solution digestion. B, Total proteins for both unique peptides 1 and 2, and coverage of proteoglycan and other ECM related proteins as observed for complex mixtures- mouse brain tissue lysate. ).

Article Snippet: R&D systems) Mouse brain tissue lysate (89-014-501, Abnova) Human neurocan recombinant protein (6508NC050, R&D systems) Aggrecan from bovine articular cartilage (A1960-1MG, SigmaAldrich) Human Syndecan-1 Protein (#7879, BioVision Incorporated) α1-Acid Glycoprotein from human plasma (#G9885, SigmaAldrich) Pierce retention time calibration mixture (#88321, ThermoScientific) Pierce™ HeLa Protein Digest Standard (#88328, Thermo Scientific) Heparan sulfate from porcine intestinal mucosa (HSPIM) (Celsus Laboratories, Inc.0 Heparan sulfate from bovine kidney (HSBK) (SigmaAldrich)

Techniques:

Journal: Molecular omics

Article Title: Serial In-solution digestion protocol for mass spectrometry-based glycomics and proteomics analysis

doi: 10.1039/d0mo00019a

Figure Lengend Snippet: Average normalized area (normalized to total ion chromatogram (TIC)) for batch 01 and 02 ± standard deviation and % relative standard deviation (%RSD) for the standard proteins.

Article Snippet: R&D systems) Mouse brain tissue lysate (89-014-501, Abnova) Human neurocan recombinant protein (6508NC050, R&D systems) Aggrecan from bovine articular cartilage (A1960-1MG, SigmaAldrich) Human Syndecan-1 Protein (#7879, BioVision Incorporated) α1-Acid Glycoprotein from human plasma (#G9885, SigmaAldrich) Pierce retention time calibration mixture (#88321, ThermoScientific) Pierce™ HeLa Protein Digest Standard (#88328, Thermo Scientific) Heparan sulfate from porcine intestinal mucosa (HSPIM) (Celsus Laboratories, Inc.0 Heparan sulfate from bovine kidney (HSBK) (SigmaAldrich)

Techniques: Standard Deviation

Journal: The Journal of Neuroscience

Article Title: CNS Progenitor Cells Promote a Permissive Environment for Neurite Outgrowth via a Matrix Metalloproteinase-2-Dependent Mechanism

doi: 10.1523/JNEUROSCI.0200-07.2007

Figure Lengend Snippet: Degradation of CD44 and neurocan by MMP2 in vitro. A–D, Incubation of rd1 retinas with recombinant MMP2 for 12 h led to a decrease in CD44 (A) and neurocan (B) immunoreactivity on the outer surface of rd1 retinas (arrowheads) compared with controls. However, the treatment did not appreciably change GFAP immunoreactivity in reactive Müller cells (C) or significantly alter retinal morphology (D). E, F, Western blot shows CD44 (E) and neurocan (F) cleavage products in rd1 explants treated with conditioned medium from rd1 retina/RPC cocultures. Lanes 1–3, Protein was extracted in culture medium from rd1 explants treated with conditioned medium from RPC (lane 1), rd1 retina/RPC cocultures (lane 2), or rd1 retina/NPC cocultures (lane 3). Lanes 4, 5, Protein was extracted from RPC (lane 4) or NPC lysate (lane 5). Scale bars: A–C, 30 μm; D, 200 μm.

Article Snippet: The primary antibodies used were raised against GFP (1:100; Sigma), CD44 (1:100; Sigma), neurocan (1:500; Renee K. Margolis, Department of Pharmacology, State University of New York, Brooklyn, NY), MMP2 (1:50; Chemicon, Temecula, CA), MMP14 (1:100; Chemicon), PKC (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA), nestin (1:1; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), neurofilament 200 (1:1000; Sigma), calbindin (D-28K, 1:800; Sigma), calretinin (1:100; Chemicon), rhodopsin (2D4, 1:500; gift from Dr. Robert Molday, University of British Columbia, Vancouver, British Columbia, Canada; 1:500), recoverin (1:200; Chemicon), cellular retinaldehyde-binding protein (1:500; Chemicon), and GFAP (1:400; Invitrogen).

Techniques: In Vitro, Incubation, Recombinant, Western Blot

Journal: The Journal of Neuroscience

Article Title: CNS Progenitor Cells Promote a Permissive Environment for Neurite Outgrowth via a Matrix Metalloproteinase-2-Dependent Mechanism

doi: 10.1523/JNEUROSCI.0200-07.2007

Figure Lengend Snippet: Reduction of CD44 and neurocan in rd1 retinas after engraftment with progenitor cells in vivo. CD44 (A) and neurocan (B) immunoreactivity was greatly reduced (arrowheads in left column) along the interface of graft and host in regions in which RPCs had migrated into the host retina at 30 d after transplantation (left column) compared with controls in which rd1 retinas were engrafted with neural retinal tissue (NR-rd1; right column). Scale bars, 30 μm.

Article Snippet: The primary antibodies used were raised against GFP (1:100; Sigma), CD44 (1:100; Sigma), neurocan (1:500; Renee K. Margolis, Department of Pharmacology, State University of New York, Brooklyn, NY), MMP2 (1:50; Chemicon, Temecula, CA), MMP14 (1:100; Chemicon), PKC (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA), nestin (1:1; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), neurofilament 200 (1:1000; Sigma), calbindin (D-28K, 1:800; Sigma), calretinin (1:100; Chemicon), rhodopsin (2D4, 1:500; gift from Dr. Robert Molday, University of British Columbia, Vancouver, British Columbia, Canada; 1:500), recoverin (1:200; Chemicon), cellular retinaldehyde-binding protein (1:500; Chemicon), and GFAP (1:400; Invitrogen).

Techniques: In Vivo, Transplantation Assay

Journal: Oncotarget

Article Title: Neurocan, an extracellular chondroitin sulfate proteoglycan, stimulates neuroblastoma cells to promote malignant phenotypes

doi: 10.18632/oncotarget.22435

Figure Lengend Snippet: ( A ) Kaplan-Meier overall survival curves based on the mRNA expression level of NCAN and other CSPGs in NB patients using scan as cut-off modus. High- and low-expression groups are indicated. The p -values were corrected for multiple testing (Bonferroni correction). n.s.: not significant. ( B ) NCAN mRNA expression levels in various cancer cell lines. ( C ) Box plot with circles indicating NCAN expression for individuals according to MYCN status, risk and INSS classifications in the Tumor NB public public-SEQC/RPM-498 dataset from R2 ( http://r2.amc.nl ). ( D ) IHC staining of NCAN in tumors of clinical patients at different stages of NB. Scale bar: 100 μm. Patients’ information was shown in .

Article Snippet: Chondroitinase ABC (chABC) and human recombinant NCAN were purchased from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Immunohistochemistry

Journal: Oncotarget

Article Title: Neurocan, an extracellular chondroitin sulfate proteoglycan, stimulates neuroblastoma cells to promote malignant phenotypes

doi: 10.18632/oncotarget.22435

Figure Lengend Snippet: ( A ) Heatmap of the relative mRNA expression levels of CSPGs based on the microarray analyses of TH -MYCN mice. ( B ) The relative mRNA expressions of NCAN, VCAN, PTPRZ1, CD44, CSPG5, TH and human MYCN in the SMG of 2-week-old WT mice and TH -MYCN hemizygous mice as determined by qPCR ( n = 5). * p < 0.01. ( C ) Hematoxylin and eosin (H&E) and IHC staining of NCAN in an SMG and tumor of a TH -MYCN mouse. Normal ganglion cells show large and slightly stained nuclei ( arrowheads ). Tumorigenic neuroblasts show small, round and deeply stained nuclei ( arrows ). Scale bar: 500 μm. ( D ) In situ hybridization of NCAN in the SMG of 2-week-old WT and TH -MYCN hemizygous mice. The tumorigenic neuroblasts were NCAN-positive ( arrows ). The boxed regions in the middle panels are magnified in the lower panels. Scale bar: 100 μm ( upper and middle ), 50 μm ( lower ). ( E ) In situ hybridization of NCAN in the terminal tumor of TH -MYCN hemizygous mice. AS: antisense, S: sense. Scale bar: 100 μm.

Article Snippet: Chondroitinase ABC (chABC) and human recombinant NCAN were purchased from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Microarray, Immunohistochemistry, Staining, In Situ Hybridization

Journal: Oncotarget

Article Title: Neurocan, an extracellular chondroitin sulfate proteoglycan, stimulates neuroblastoma cells to promote malignant phenotypes

doi: 10.18632/oncotarget.22435

Figure Lengend Snippet: (A) Western blot for NCAN expression in mouse (Neuro2a) and human NB cell lines. Endogenous full-length NCAN is indicated by the arrowhead. ( B) Representative fluorescent images of venus-expressing adherent cells and NCAN-expressing sphere cells derived from YT-nu, TNB1 and NB39 cells. The pictures were taken 72 hr after infection with lentivirus overexpressing either venus or NCAN. Scale bars: 100 μm. ( C) Western blot for NCAN expressed and secreted from the cells shown in panel B. The mediums were harvested and then untreated or treated with chABC at 37°C for 1 hr. ( D) The anchorage-independent colony formation assay. Representative crystal violet-staining images and the colony numbers (dia. >200 μm) in soft agar are shown ( n = 3). * p < 0.001. ( E) The subcutaneous xenografts in KSN/Slc nude mice. The image of tumors derived from either venus- or NCAN-expressing NB39 cells ( left ), quantified tumor volumes ( middle ), and weights ( right ). Tumors were dissected at 4 weeks after inoculation. * p < 0.001. Scale bars: 1 cm.

Article Snippet: Chondroitinase ABC (chABC) and human recombinant NCAN were purchased from R&D Systems (Minneapolis, MN).

Techniques: Western Blot, Expressing, Derivative Assay, Infection, Colony Assay, Staining

Journal: Oncotarget

Article Title: Neurocan, an extracellular chondroitin sulfate proteoglycan, stimulates neuroblastoma cells to promote malignant phenotypes

doi: 10.18632/oncotarget.22435

Figure Lengend Snippet: (A) TNB1 cells were cultured for 2 days in serum-free medium containing 10 μg/ml of recombinant NCAN. The boxed region is magnified at the right. Scale bars: 100 μm. ( B) The treatment of TNB1 cells with conditioned medium containing overexpressed and secreted NCAN from YT-nu cells. The conditioned medium of venus-expressing YT-nu cells was prepared as a control. The conditioned media were either untreated or treated with chABC. Representative images of TNB1 cells treated with each conditioned medium are shown. Scale bars: 100 μm. ( C) TNB1 cells were treated with conditioned medium whose NCAN core protein was heat-denatured (boiled for 15 min). Representative pictures ( left: control, right: heat-denatured) are shown. Scale bars: 100 μm. ( D) TNB1 cells were treated with conditioned medium whose proteins were digested by trypsin. The conditioned media were digested by 0.01% of trypsin (37°C for 15 min), followed by the addition of FBS to inactivate trypsin. Representative pictures ( left: control, right: trypsinized) are shown. The boxed region is magnified in the middle panel. Scale bars: 100 μm. ( E) Schematic of full-length NCAN and five other truncated versions of NCAN. NCAN consists of G1, CS and G3 domains. Every version of NCAN is fused with AP at the N-terminal. sp: signal peptide. ( F) Western blot of each conditioned medium with anti-NCAN antibody. The media were treated or untreated with chABC. ( G) Western blot of each conditioned medium with anti-AP antibody. The media were treated or untreated with chABC. ( H) A series of AP-fused NCAN-containing conditioned media were examined for their ability to induce the sphere formation of TNB1 cells.

Article Snippet: Chondroitinase ABC (chABC) and human recombinant NCAN were purchased from R&D Systems (Minneapolis, MN).

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

Journal: Oncotarget

Article Title: Neurocan, an extracellular chondroitin sulfate proteoglycan, stimulates neuroblastoma cells to promote malignant phenotypes

doi: 10.18632/oncotarget.22435

Figure Lengend Snippet: (A) Results of the GO analysis of upregulated ( left ) or downregulated genes ( right ) in NB39 cells treated with NCAN-containing conditioned medium compared with those treated with venus-containing conditioned medium. The top 20 GO biological process terms based on fold enrichment are shown. ( B) The GSEA results indicated that the gene set related to the cell cycle was significantly enriched in NB39 cells treated with NCAN-containing conditioned medium. ( C) The RT-qPCR results of cyclin-dependent kinases (CDKs) and CDK inhibitors in NB39 cells treated with NCAN-containing conditioned medium compared with those treated with venus-containing conditioned medium. * p < 0.01. ( D) The results of the RT-qPCR of differentiation and stemness marker genes in NB39 cells treated with NCAN-containing conditioned medium compared with those treated with venus-containing conditioned medium. * p < 0.01. ( E) The RT-qPCR of the differentiation and stemness marker genes in TNB1 cells infected with NCAN-expressing or venus-expressing lentivirus. * p < 0.01. ( F) The GSEA indicated that the set of MYC targets was significantly enriched in NB39 cells treated with NCAN-containing conditioned medium.

Article Snippet: Chondroitinase ABC (chABC) and human recombinant NCAN were purchased from R&D Systems (Minneapolis, MN).

Techniques: Quantitative RT-PCR, Marker, Infection, Expressing

Journal: Oncotarget

Article Title: Neurocan, an extracellular chondroitin sulfate proteoglycan, stimulates neuroblastoma cells to promote malignant phenotypes

doi: 10.18632/oncotarget.22435

Figure Lengend Snippet: ( A ) RT-qPCR revealed the efficient knockdown of NCAN mRNA by two independent shRNAs in the tumor sphere line #6673 derived from TH -MYCN mice. * p < 0.05, ** p < 0.01. ( B ) Western blot for mouse endogenous NCAN showing the efficient knockdown by shRNAs in the tumor sphere line #6673 derived from TH -MYCN mice. ( C ) Schematic of NCAN knockdown in mouse tumor sphere cells. ( D ) Representative images of tumor spheres after the knockdown of NCAN. Day 4: Four days after infection with shRNA-expressing lentivirus. Day 8: Just before the quantification of spheres. Scale bars: 100 μm. ( E ) Quantification of the sphere numbers and sizes shown in panel C. ** p < 0.01. ( F ) RT-qPCR analysis of stemness marker genes in each tumor sphere. ** p < 0.01. (G ) Here, 1 × 10 5 tumor sphere cells expressing each shRNA were subcutaneously inoculated into KSN/Slc nude mice. The tumor forming ratio is indicated ( n = 6 for shRNA-NT, n = 8 for shRNA#59 and shRNA#60).

Article Snippet: Chondroitinase ABC (chABC) and human recombinant NCAN were purchased from R&D Systems (Minneapolis, MN).

Techniques: Quantitative RT-PCR, Knockdown, Derivative Assay, Western Blot, Infection, shRNA, Expressing, Marker

Journal: Scientific Reports

Article Title: Single-nucleus transcriptomic profiling of the diaphragm during mechanical ventilation

doi: 10.1038/s41598-024-82530-4

Figure Lengend Snippet: Differentially expressed genes and Kyoto Encyclopedia of Genes and Genomes analysis. Volcano plots showing the top twenty up- or downregulated genes for each cell type. The red dots represent upregulated genes, and the blue dots represent downregulated genes. ( P value < 0.05, and |log2foldchange| > 0.58). Bubble plot indicating the top enriched pathways for each cell type based on KEGGpathway enrichment analysis of differentially expressed genes( www.kegg.jp/kegg/kegg1.html ). The sizes of the dots represent the number of genes included in each pathway. The colour gradient of dots represents the adjusted P values of each enriched pathway. The genes Pfkfb3 , Tbc1d1 and the insulin signaling pathway; Pdgfd and the PI3K-Akt signaling pathway; Cxcr2 and PLD, Rap1 signaling pathways; Ccl21 and chemical carcinogenesis-reactive oxygen species signaling pathway; Mef2c and the calcium signaling pathway; Negr1 and leukocyte transendothelial migration signaling pathway are labelled by the black and red bars in Panels a , b , c , d , e , and f .

Article Snippet: After the semidry blotting procedure (50 min, 90 V), the membrane was incubated for 1 h at room temperature (RT) in 5% BSA blocking solution, followed by overnight incubation on a shaker at 4 °C with primary antibodies against PFKFB3 (bs-3528R, Boster Biological Technology Co., Ltd., Wuhan, China), PDGFD (bs-24572R, Boster), CXCR2 (bs-1629R, Boster), NEGR1 (bs-11095R, Boster), SEMA3A (bs-10468R, Boster), MEF2C (bs-4130R, Boster) and β-actin (66009-1-Ig, Proteintech, Wuhan, China) as a loading control.

Techniques: Protein-Protein interactions, Migration

Journal: Scientific Reports

Article Title: Single-nucleus transcriptomic profiling of the diaphragm during mechanical ventilation

doi: 10.1038/s41598-024-82530-4

Figure Lengend Snippet: Protein-protein interaction network (PPI network).We constructed nine PPI networks displaying protein-protein interactions among the related genes. The nodes represent proteins, and the edges represent the interaction strength between two proteins. The proteins PFKFB3 ( a ), PDGFD ( b ), CXCR2 ( c ), CCL21 ( d ), SEMA3A ( e ), RYR3 ( f ), MEF2C ( g ), NEGR1( h ) and TBC1D1 ( i ) which are located at the hub of the interaction network, are responsible for diaphragm fibrosis and atrophy.

Article Snippet: After the semidry blotting procedure (50 min, 90 V), the membrane was incubated for 1 h at room temperature (RT) in 5% BSA blocking solution, followed by overnight incubation on a shaker at 4 °C with primary antibodies against PFKFB3 (bs-3528R, Boster Biological Technology Co., Ltd., Wuhan, China), PDGFD (bs-24572R, Boster), CXCR2 (bs-1629R, Boster), NEGR1 (bs-11095R, Boster), SEMA3A (bs-10468R, Boster), MEF2C (bs-4130R, Boster) and β-actin (66009-1-Ig, Proteintech, Wuhan, China) as a loading control.

Techniques: Construct, Protein-Protein interactions

Journal: Scientific Reports

Article Title: Single-nucleus transcriptomic profiling of the diaphragm during mechanical ventilation

doi: 10.1038/s41598-024-82530-4

Figure Lengend Snippet: Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting. qRT-PCR and Western blotting showing the expression of selected genes and corresponding proteins in the mechanically ventilated diaphragm and control ones. The bar graphs show the quantification of the mRNA expression of Pfkfb3 ( a ), Pdgfd ( b ), Cxcr2 ( c ), Negr1 ( d ), Sema3a ( e ), and Mef2c ( f ) normalized to that of GAPDH. Asterisks indicate significant differences ( n = 3 in each group) (* P < 0.05). Western blotting analysis of the protein expression of PFKFB3 ( g ), PDGFD ( h ), CXCR2 ( i ), NEGR1 ( j ), SEMA3A ( k ), and MEF2C ( l ) normalized to that of β-actin as a loading control.

Article Snippet: After the semidry blotting procedure (50 min, 90 V), the membrane was incubated for 1 h at room temperature (RT) in 5% BSA blocking solution, followed by overnight incubation on a shaker at 4 °C with primary antibodies against PFKFB3 (bs-3528R, Boster Biological Technology Co., Ltd., Wuhan, China), PDGFD (bs-24572R, Boster), CXCR2 (bs-1629R, Boster), NEGR1 (bs-11095R, Boster), SEMA3A (bs-10468R, Boster), MEF2C (bs-4130R, Boster) and β-actin (66009-1-Ig, Proteintech, Wuhan, China) as a loading control.

Techniques: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Expressing, Control

Journal: Molecular Neurobiology

Article Title: Runx2 Suppresses Astrocyte Activation and Astroglial Scar Formation After Spinal Cord Injury in Mice

doi: 10.1007/s12035-024-04212-6

Figure Lengend Snippet: Overexpressing Runx2 inhibited the activation of astrocytes following SCI in vivo. A Western blot analysis of Neurocan (143 kDa) and GFAP (55 kDa)in lesions, with GAPDH (37 kDa) as an internal loading control. B , C Quantification of result in panel ( A ). The data were analyzed using one-way analysis of variance and all data are expressed as the mean ± S.D. D Immunofluorescence staining of DAPI (blue), GFAP (red), and Runx2 (green) in the spinal cord at 14 days post-injury. * p < 0.05; ** p < 0.01; *** p < 0.001. Scale bar = 200 μm. Runx2-OE (AAV-Runx2)

Article Snippet: Then incubate overnight at 4 °C with the following primary antibodies: Runx2 (1:1000; Abcam); GFAP (1:1000; Abcam); polyclonal GAPDH antibody (1:1000; Proteintech); polyclonal S100A10 antibody (1:1000; Proteintech); Neurocan (1:1000; Zenbio); and NF-κB p65 (1:1000; Proteintech).

Techniques: Activation Assay, In Vivo, Western Blot, Control, Immunofluorescence, Staining

Journal: Molecular Neurobiology

Article Title: Runx2 Suppresses Astrocyte Activation and Astroglial Scar Formation After Spinal Cord Injury in Mice

doi: 10.1007/s12035-024-04212-6

Figure Lengend Snippet: The effect of Runx2 nuclear import is crucial on modulation of astroglial scar. A Western blot analysis of Runx2 in nucleus (57 kDa) and GFAP (55 kDa), with GAPDH (37 kDa) as an internal loading control. B , C Quantification of result in panel ( A ). D Immunofluorescence staining of DAPI (blue), GFAP (red), and Runx2 (green) in the SVG-P12. E Immunofluorescence staining of DAPI (blue), Neurocan (red), and Runx2 (green) in the SVG-P12. The data were analyzed using one‑way analysis of variance, and all data are expressed as the mean ± S.D. * p < 0.05; ** p < 0.01; *** p < 0.001. Scale bar = 20 μm. DC (plasmid without Runx2), DR (plasmid of Runx2 with NMTS), DM (plasmid of Runx2 without NMTS)

Article Snippet: Then incubate overnight at 4 °C with the following primary antibodies: Runx2 (1:1000; Abcam); GFAP (1:1000; Abcam); polyclonal GAPDH antibody (1:1000; Proteintech); polyclonal S100A10 antibody (1:1000; Proteintech); Neurocan (1:1000; Zenbio); and NF-κB p65 (1:1000; Proteintech).

Techniques: Western Blot, Control, Immunofluorescence, Staining, Plasmid Preparation