Journal: Frontiers in Cell and Developmental Biology

Article Title: Unraveling the impact of human cerebrospinal fluid on human neural stem cell fate

doi: 10.3389/fcell.2025.1527557

Figure Lengend Snippet: Primary antibodies used for IF staining.

Article Snippet: NG2 , Rabbit polyclonal , IgG , 1:150 , Millipore , AB5320.

Techniques: Staining

Journal: iScience

Article Title: Inhibitory maturation and ocular dominance plasticity in mouse visual cortex require astrocyte CB1 receptors

doi: 10.1016/j.isci.2024.111410

Figure Lengend Snippet: Early astrocytic recombination in GLAST-CreERT2 mice (A) GLAST-CreERT2 TdTomato mice received a single i.p. injection of tamoxifen at P1. Slices containing V1 were prepared at P28-35, processed for immunofluorescence imaging and visualized using confocal microscopy. Recombination (indicated by TdTomato expression, magenta) was observed in astrocytes (visualized using a glutamine synthetase antibody, green) but also in sparse neurons (visualized using NeuN antibody, blue). An example neuron with pyramidal morphology is indicated by the arrow. Scale bar, 100 μm. (B) Changing the tamoxifen injection regime to a single injection at P3-5 abolished neuronal recombination in V1, while astrocyte recombination was efficient (∼80%). Arrows indicate TdTomato-expressing astrocytes, arrowheads indicate TdTomato-negative astrocytes. (C and D) Specificity of recombination was high for astrocytes, but some non-neuronal recombination was seen in glial cells positive for NG2 (C, green, arrows) or Olig2 (D, green, arrows). Arrowheads indicate TdTomato-negative NG2-positive and Olig2-positive cells. (E) Quantification of efficiency and specificity of recombination in astrocytes in mice receiving a single tamoxifen i.p. injection at P3-5, based on TdTomato and glutamine synthetase positivity ( N = 6). Error bars indicate SEM.

Article Snippet: NG2 (polyclonal rabbit) , Merck Millipore , AB5320; RRID: AB_91789.

Techniques: Injection, Immunofluorescence, Imaging, Confocal Microscopy, Expressing

Journal: iScience

Article Title: Inhibitory maturation and ocular dominance plasticity in mouse visual cortex require astrocyte CB1 receptors

doi: 10.1016/j.isci.2024.111410

Figure Lengend Snippet:

Article Snippet: NG2 (polyclonal rabbit) , Merck Millipore , AB5320; RRID: AB_91789.

Techniques: Recombinant, Ointment, Software

Journal: Life Science Alliance

Article Title: Lineage-specific changes in mitochondrial properties during neural stem cell differentiation

doi: 10.26508/lsa.202302473

Figure Lengend Snippet: (A, B) Representative confocal digital images of mitochondrial network in oligodendrocyte precursor cells (PDGFRα+/NG2+) (A) and oligodendrocytes (MBP+) at different stages of maturation (B). Scale bar: 30 μm.

Article Snippet: Rabbit polyclonal Chondroitin Sulphate Proteoglycan NG2 , Millipore , Cat# AB5320.

Techniques:

Journal: Life Science Alliance

Article Title: Lineage-specific changes in mitochondrial properties during neural stem cell differentiation

doi: 10.26508/lsa.202302473

Figure Lengend Snippet: List of materials.

Article Snippet: Rabbit polyclonal Chondroitin Sulphate Proteoglycan NG2 , Millipore , Cat# AB5320.

Techniques: Magnetic Beads, Isolation, Detection Assay, Software

Journal: iScience

Article Title: Reduced chondroitin sulfate content prevents diabetic neuropathy through transforming growth factor-β signaling suppression

doi: 10.1016/j.isci.2024.109528

Figure Lengend Snippet: BNB integrity against hazardous stimuli was maintained in T1KO mice (A) Representative images of sciatic nerves injected with 70 kDa FITC-dextran in the absence and presence of hyperglycemic stimuli. Arrows indicate extra-vascular leakage of FITC-dextran. Note that the WT-STZ group, but not the T1KO-STZ group, showed leakage from the vessels. (B) Representative IHC images of sciatic nerve cross-sections. BNB was defined as a structure in which PDGFRβ (a pericyte marker)-positive cells surround CD31 (an endothelial cell marker)-positive cells. Red : CD31; Green : PDGFRβ. A rrows ; BNB structures. The number of BNBs per sciatic nerve (C) and (D) per area at three weeks after STZ injection. Mean ± SD is shown, n = 3 per group. (E) Representative IHC images of retinal vessels on postnatal day 8 (P8) after anti-PDGFRβ Ab injection. Green : CD31; Red : NG2. (F–H) Quantification of retinal vessel diameters after anti-PDGFRβ Ab administration, compared to the control groups; (F) arteries, (G) capillaries, and (H) veins. (I and J) Measurement of CD31-positive areas (I) and NG2 coverage (J) in P8 retina after anti-PDGFRβ Ab administration, compared to the control groups. Mean ± SD is shown with ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001 for comparison, one-way ANOVA with Tukey’s multiple comparisons test, n = 3 per group.

Article Snippet: NG2 (Rabbit Polyclonal anti-NG2 antibody) , Merck Millipore , AB5320.

Techniques: Injection, Marker, Control, Comparison

Journal: iScience

Article Title: Reduced chondroitin sulfate content prevents diabetic neuropathy through transforming growth factor-β signaling suppression

doi: 10.1016/j.isci.2024.109528

Figure Lengend Snippet:

Article Snippet: NG2 (Rabbit Polyclonal anti-NG2 antibody) , Merck Millipore , AB5320.

Techniques: Purification, Recombinant, TaqMan Assay, Reverse Transcription, Software

Journal: The Journal of Biological Chemistry

Article Title: Chondroitin sulfate modification of CSPG4 regulates the maintenance and differentiation of glioma-initiating cells via integrin-associated signaling

doi: 10.1016/j.jbc.2024.105706

Figure Lengend Snippet: CSPG4–chondroitin sulfate proteoglycan chains suppress GIC differentiation. Cell morphology of GICs treated with chABC and heparinase I/III and II. GICs cultured in serum-free medium were treated with 0.05 U/ml of chABC, heparinase I/III, or heparinase II, followed by observation of cell morphology by microscopy after 48 h. Scale bars represent 100 μm. B , expression of CSPG4–CS and -core in GICs treated with chABC. GFAP was used as a differentiation marker. GICs were incubated with chABC for 48 h, followed by Western blot analysis. Arrows indicate CS-modified CSPG4 ( top ) and nonglycosylated core CSPG4 ( bottom ). C , morphology of serum-induced differentiating cells treated with or without chABC. GICs induced to differentiate in 1% serum were cultured with chABC, and morphological changes were observed after 24 h. Scale bars represent 100 μm. D , expression of CSPG4 and GFAP in serum-induced differentiating GICs with and without chABC. GICs were induced to differentiate in 1% serum and treated with or without chABC for 24 h. Cells were lysed and followed by Western blot analysis. E and F , the morphology of chABC- ( E ) or serum- ( F ) induced differentiating cells treated with 500 μg/ml of CS-AC or CS-B, respectively. Serum concentrations were used at 1% for GIC03A and 2% for GIC03U. Scale bars represent 100 μm ( E ) and 50 μm ( F ). chABC, chondroitinaseABC; CSPG4, chondroitin sulfate proteoglycan 4; GFAP, glial fibrillary acid protein; GIC, glioma-initiating cell.

Article Snippet: For CSPG4 and CS staining, cells were fixed with 4% paraformaldehyde, permeabilized, blocked with 5% bovine serum albumin, and reacted with primary antibodies: mouse monoclonal anti-CS proteoglycan (CSPG) antibody (Millipore), rabbit polyclonal anti-NG2 CS proteoglycan antibody (Millipore), and mouse monoclonal anti-CS antibody (Abcam) as described previously.

Techniques: Cell Culture, Microscopy, Expressing, Marker, Incubation, Western Blot, Modification

Journal: The Journal of Biological Chemistry

Article Title: Chondroitin sulfate modification of CSPG4 regulates the maintenance and differentiation of glioma-initiating cells via integrin-associated signaling

doi: 10.1016/j.jbc.2024.105706

Figure Lengend Snippet: CSPG4 regulates GIC differentiation. A , cell morphology of GICs transfected with siControl or siCSPG4 and induced to differentiate in chABC or serum. GICs were transfected with siControl or siCSPG4, cultured for 24 h, and then treated with chABC or 1% serum for 48 h. Scale bars represent 50 μm. B and C , effects of CSPG4 knockdown against GIC cell differentiation. After transfection with CSPG4 siRNA (siCSPG4) or control siRNA (sicont), GICs were cultured in serum-free medium for 24 h and then treated with chABC ( B ) or 1% serum ( C ) for 48 h. Cells were lysed and subjected to GFAP expression analysis. GFAP was quantified and normalized with β-actin, presented as the mean ± SD of three independent experiments. Significance was tested by multicomparison test. C , GIC03U: ∗∗∗ p = 0.0004. ChABC, chondroitinaseABC; CSPG4, chondroitin sulfate proteoglycan 4; GFAP, glial fibrillary acid protein; GIC, glioma-initiating cell.

Article Snippet: For CSPG4 and CS staining, cells were fixed with 4% paraformaldehyde, permeabilized, blocked with 5% bovine serum albumin, and reacted with primary antibodies: mouse monoclonal anti-CS proteoglycan (CSPG) antibody (Millipore), rabbit polyclonal anti-NG2 CS proteoglycan antibody (Millipore), and mouse monoclonal anti-CS antibody (Abcam) as described previously.

Techniques: Transfection, Cell Culture, Knockdown, Cell Differentiation, Control, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Chondroitin sulfate modification of CSPG4 regulates the maintenance and differentiation of glioma-initiating cells via integrin-associated signaling

doi: 10.1016/j.jbc.2024.105706

Figure Lengend Snippet: CSPG4–integrin interaction induced by chABC upregulates ITGAV -dependent ERK/MAPK signaling. A , cell morphology of GICs and chABC-induced differentiating cells with or without cRGD or CS. Cells were cultured with 300 μM cRGD peptide or 500 μg/ml CS-A/C for 30 min followed by the treatment with or without chABC for 24 h. Scale bars represent 100 μm. B , phospho-ERK activation in GICs and chABC-induced differentiating cells. GICs were incubated with 500 μM cRGD peptide for 30 min and subsequently cultured with chABC for 48 h. pERK were analyzed by Western blot. C , effects of cRGD on SOX2 and phospo-ERK in GICs and chABC-induced differentiating cells. GICs and chABC-induced differentiating cells were cultured with or without cRGD peptide for 24 h. Fixed cells were stained with Alexa Fluor-488 ( green ) for SOX2 as a GIC marker and Alexa Fluor-546 ( red ) for pERK. Scale bars represent 100 μm. D , immunoprecipitation analysis to determine the binding between CSPG4 and ITGAV. Lysates from GICs or chABC-induced differentiating cells were subjected to immunoprecipitation using anti-CSPG4 antibody, followed by immunoblotting with anti-CSPG4, anti-ITGAV, or anti-integrin α5 antibodies ( left ). Whole-cell lysates (input) were analyzed by immunoblotting with antibodies against CSPG4, ITGAV, integrin α5, GFAP, and β-actin to assess each protein expression level ( right ). Black arrowheads showed CS-modified form of CSPG4 (CS) and a nonglycosylated core form of CSPG4 (core). Red arrowheads indicated the binding of ITGAV and CSPG4 in chABC-induced differentiating cells. ERK, extracellular signal–regulated kinase; chABC, chondroitinaseABC; cRGD, cyclic-Arg-Gly-Asp; CS, chondroitin sulfate; CSPG4, chondroitin sulfate proteoglycan 4; GIC, glioma-initiating cell; ITGAV, integrin αV; MAPK, mitogen-activated protein kinase; pERK, phospho-ERK.

Article Snippet: For CSPG4 and CS staining, cells were fixed with 4% paraformaldehyde, permeabilized, blocked with 5% bovine serum albumin, and reacted with primary antibodies: mouse monoclonal anti-CS proteoglycan (CSPG) antibody (Millipore), rabbit polyclonal anti-NG2 CS proteoglycan antibody (Millipore), and mouse monoclonal anti-CS antibody (Abcam) as described previously.

Techniques: Cell Culture, Activation Assay, Incubation, Western Blot, Staining, Marker, Immunoprecipitation, Binding Assay, Expressing, Modification

Journal: The Journal of Biological Chemistry

Article Title: Chondroitin sulfate modification of CSPG4 regulates the maintenance and differentiation of glioma-initiating cells via integrin-associated signaling

doi: 10.1016/j.jbc.2024.105706

Figure Lengend Snippet: GBMs with high CSPG4 expression have correlations with XYLT1 and a poor prognosis of patients. A , differential analysis of CSPG4 expression in tumors (num [T] = 163 samples) and normal samples (num [N] = 207 samples) using GBM transcriptome datasets in GEPIA. B , overall survival of GBM patients with high and low CSPG4 expression in GBM patients. C , correlation between CSPG4 and XYLT1 expressions in GBMs. CSPG4, chondroitin sulfate proteoglycan 4; GBM, glioblastoma multiforme; GEPIA, Gene Expression Profiling Interactive Analysis; XYLT1, xylosyltransferase 1.

Article Snippet: For CSPG4 and CS staining, cells were fixed with 4% paraformaldehyde, permeabilized, blocked with 5% bovine serum albumin, and reacted with primary antibodies: mouse monoclonal anti-CS proteoglycan (CSPG) antibody (Millipore), rabbit polyclonal anti-NG2 CS proteoglycan antibody (Millipore), and mouse monoclonal anti-CS antibody (Abcam) as described previously.

Techniques: Expressing, Gene Expression

Journal: Cancers

Article Title: Inhibition of EphA3 Expression in Tumour Stromal Cells Suppresses Tumour Growth and Progression

doi: 10.3390/cancers15184646

Figure Lengend Snippet: EphA3 is expressed on cells associated with angiogenesis. ( A ) Aortic explants from mice were incubated in Matrigel and VEGF-A to stimulate vessel sprouting, and then stained with Rhodamine-lectin (marking vessels), DAPI (marking nuclei), and antibodies for EphA3 and NG2 (perivascular cell marker). Images were obtained using confocal fluorescence microscopy. A vessel sprout indicated in the low-magnification image of a sprouting aortic explant (left panel) is shown at a higher magnification (middle panels) using combined z-stack images of individual and merged channels, as indicated. Right panels: the top panel shows a single xy and z-stack series of a region showing EphA3 staining (arrows) in NG2-positive perivascular cells. White lines in images indicate planes of associated xy, xz and yz sections; the bottom panel shows three-dimensional rendering of a z-stack showing EphA3 staining at boundaries between perivascular cells and lectin-stained endothelial cells (arrows). Scale bars in microns. ( B ) Primary aortic cell cultures were stained with antibodies against EphA3 (Alexa-647 conjugated), and markers for MSCs (CD140a, CD90, Sca1), endothelial cells (CD31, VE-cadherin), and haematopoietic cells (CD45) before analysis by flow cytometry. Dot plots (left) show EphA3 staining (fluorescence versus forward scatter) compared to unstained control cells; histograms show co-staining of EphA3 positive or total viable cells, with the indicated markers or unstained cells as control.

Article Snippet: NG2 rabbit polyclonal antibody (ab5329) was from Millipore; rat anti-mouse CD90-PB (30-H12), CD45-PB (30F-11) and Sca1-PE/Cy7 (D7) monoclonal antibodies were from BioLegend, San Diego, CA, USA; rat anti-mouse CD8 (4SM15) and TER119 (ef450 conjugated 48-5921-80) monoclonal antibodies were from eBioscience (ThermoFischer, San Diego, CA, USA); CD31 rat anti-mouse monoclonal antibody (Clone MEC 13.3) was from Becton Dickinson; rabbit transgelin (155272) polyclonal and alpha smooth muscle actin (SMA) (ab124964) monoclonal antibodies were from Abcam (Cambridge, UK); α-EphA3 mouse monoclonal IIIA4 and sheep polyclonal antibodies have been previously described [ , ].

Techniques: Incubation, Staining, Marker, Fluorescence, Microscopy, Flow Cytometry, Control