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: 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