Journal: Cell Communication and Signaling : CCS

Article Title: Rho GTPases signaling mediates aggressiveness and differentiation in neuroblastoma tumors

doi: 10.1186/s12964-025-02649-3

Figure Lengend Snippet: ARHGAP31 /CdGAP is associated with undifferentiated cells, and its downregulation provokes Cdc42 overexpression. A mRNA expression of ARHGAP31 in adrenergic tumors, mesenchymal tumors or neural crest tissues. Long rank test p value is shown, * = p < 0.05. Dataset: GSE908030. B mRNA expression of ARHGAP31 in SK-N-SH CD44hi-derived undifferentiated secondary tumorspheres (TS2) compared to the bulk cell line. * = p < 0.05, n = 4. C Immunofluorescence showing CdGAP expression in NB48T CD44hi-derived undifferentiated tumorspheres compared to CD44negative-derived ones. D Tumorsphere formation efficiency of SK-N-SH or NB48T PDX-derived cell lines after CdGAP knock down by siRNA or control. * = p < 0.05; *** = p < 0.005. E Expression of the undifferentiated marker nestin in SK-N-SH and NB48T PDX-derived tumorspheres after CdGAP knock down by siRNA or control. ** = p < 0.01; **** = p < 0.001. F Expression of Cdc42 in SK-N-SH and NB48T PDX-derived TS cells after CdGAP knock down by siRNA or control. Representative images from NB48T cells are shown. **** = p < 0.001. G Expression of Sox9 protein in cells from tumorspheres generated from NB48T or sorted CD44hi SK-N-SH and NB48T cells, after CdGAP knock down by siRNA or control. * = p < 0.05; **** = p < 0.001. H Cell viability in CHLA 20 cells, with or without downregulation of CdGAP, over 72 h (** = p < 0.01). I Quantification of proliferating Ki67 expressing cells in NB48T cells with or without downregulation of CdGAP. Graph shows mean ± SEM. Scale bar = 20μm. (* = p < 0.05, n = 3). J Morphological characterization of CHLA 20 and NB48T cells with or without downregulation of CdGAP. Cell segmentation on the cell contour is shown in red. Scale bar = 20 μm. K Immunofluorescence for acetylated tubulin, and DAPI in CHLA 20 cells with or without downregulation of CdGAP. The presence of acetylated tubulin in cell extensions is indicated with red asterisks. Scale bar = 20 μm. L Quantification of the cells positive for acetylated tubulin staining in cellular protrusions after the indicated treatments in CHLA20 cells (mean ± SEM, * = p < 0.05; ** = p < 0.001, n = 3)

Article Snippet: For immunofluorescence, cells were fixed with 4% PFA in PBS for 15 min, permeabilized with 0.2% Triton X-100 in PBS for 10 min at 4 °C and blocked with PBS + 1% BSA for at least 1 h. Primary antibodies used were: CD44 mouse (1:200 BD Pharmingen), Cdc42 rabbit (1:1000 Cell Signaling), Ki67 rabbit (1:200 Thermo Scientific), Nestin rabbit (1:1000 Millipore), Nestin mouse (1:1000 R&D Systems), α-tubulin mouse (1:1000 Sigma), acetylated tubulin mouse (1:1000, Santa Cruz), Tuj1-βIII tubulin mouse (1:1000 abcam) and CdGAP (1:500, Santa Cruz).

Techniques: Over Expression, Expressing, Derivative Assay, Immunofluorescence, Knockdown, Control, Marker, Generated, Staining

Journal: Frontiers in Molecular Neuroscience

Article Title: PDGFR-β-Positive Perivascular Adventitial Cells Expressing Nestin Contribute to Fibrotic Scar Formation in the Striatum of 3-NP Intoxicated Rats

doi: 10.3389/fnmol.2018.00402

Figure Lengend Snippet: The spatiotemporal relationship of PDGFR-β and two intermediate filament proteins nestin and vimentin in the lesioned striatum at days 3 and 7 after 3-NP injection. (A,B,E,F) Triple-labeling for PDGFR-β, GFAP and nestin at day 3 post-lesion showing that vascular profiles expressing PDGFR-β are positive for nestin in the lesion core (right side of the broken line), which is devoid of GFAP immunoreactivity. Note that nestin-positive cells in the peri-lesional area are astrocytes. (C,D,G,H) At 7 days, distribution of PDGFR-β in the vascular profiles overlaps well with that of nestin in the lesion core (right side of the broken line). (E–H) Higher magnification images of the boxed areas in (A–D) , respectively. (I–K) Triple-labeling for nestin, Ki-67 and GFAP. Note that a pair of nestin-positive cells that appear to be daughter cells are positive for Ki-67. (L) 5-Bromo-2-deoxyuridine (BrdU) injection protocol. (M,N) Triple-labeling for nestin, BrdU and GFAP showing that PDGFR-β/nestin double-positive cells within vascular profiles exhibit BrdU-labeled cell nuclei. The boxed area in (M) is enlarged in (N) . (O–R) Triple labeling for PDGFR-β, nestin and vimentin showing that nearly all of PDGFR-β/nestin double-positive cells co-express vimentin. Cell nuclei are stained with DAPI. Scale bars = 100 μm for (A–D) ; 20 μm for (E–H,I,J–M) ; 10 μm for (N) ; 50 μm for (O–R) .

Article Snippet: Primary antibodies and dilutions were as follows: rabbit monoclonal antibody against PDGFR-β (1:200; Abcam), mouse monoclonal antibody against RECA1 (1:200; Bio-Rad, Hercules, CA, USA), chicken polyclonal antibody against glial fibrillary acidic protein (GFAP; 1:500; Millipore), goat polyclonal antibody against type IV collagen (1:100; Bio-Rad), mouse monoclonal antibody against nestin (1:500; Bio-Rad), goat polyclonal antibody against ionized calcium-binding adaptor molecule 1 (Iba1; 1:500; Abcam), chicken polyclonal antibody against vimentin (1:500, Millipore), mouse monoclonal antibody to NG2 (1:500; Millipore), or rabbit polyclonal antibody against Ki-67 (1:1,000; Leica Biosystems, Wetzlar, Germany).

Techniques: Injection, Labeling, Expressing, Staining

Journal: Frontiers in Molecular Neuroscience

Article Title: PDGFR-β-Positive Perivascular Adventitial Cells Expressing Nestin Contribute to Fibrotic Scar Formation in the Striatum of 3-NP Intoxicated Rats

doi: 10.3389/fnmol.2018.00402

Figure Lengend Snippet: Characterization of extravascular PDGFR-β-positive cells in the lesioned striatum at days 14 and 28 after 3-NP injection. (A–H) Triple labeling for PDGFR-β, GFAP and nestin at day 14 (A,C–E) and 28 (B,F–H) post-lesion showing that the distribution of PDGFR-β overlaps well with that of nestin. Note that PDGFR-β-positive processes show finer intertwined profiles, while nestin staining reveals distinct fibrous structures. (C–H) Higher magnification images of the boxed areas in (A,B) , respectively. (I–N) Double labeling for nestin and either laminin (I–K) or fibronectin (L–N) , showing that nestin is colocalized with two stromal cell markers. (O,Q–T) Triple labeling for PDGFR-β, nestin and collagen IV showing that expression of the three proteins generally overlap in the lesion core. The boxed area in (O) is enlarged in (Q–T) . (P) Histogram of the intensity profiles of PDGFR-β, nestin and collagen IV along the indicated area (white arrows in Q–S ) showing that the three signals share overlapping profiles. Cell nuclei are stained with DAPI. Scale bars = 100 μm for (A,B,O) ; 20 μm for (I–N„Q–T) ; 10 μm for (C–H) .

Article Snippet: Primary antibodies and dilutions were as follows: rabbit monoclonal antibody against PDGFR-β (1:200; Abcam), mouse monoclonal antibody against RECA1 (1:200; Bio-Rad, Hercules, CA, USA), chicken polyclonal antibody against glial fibrillary acidic protein (GFAP; 1:500; Millipore), goat polyclonal antibody against type IV collagen (1:100; Bio-Rad), mouse monoclonal antibody against nestin (1:500; Bio-Rad), goat polyclonal antibody against ionized calcium-binding adaptor molecule 1 (Iba1; 1:500; Abcam), chicken polyclonal antibody against vimentin (1:500, Millipore), mouse monoclonal antibody to NG2 (1:500; Millipore), or rabbit polyclonal antibody against Ki-67 (1:1,000; Leica Biosystems, Wetzlar, Germany).

Techniques: Injection, Labeling, Staining, Expressing

Journal: Frontiers in Molecular Neuroscience

Article Title: PDGFR-β-Positive Perivascular Adventitial Cells Expressing Nestin Contribute to Fibrotic Scar Formation in the Striatum of 3-NP Intoxicated Rats

doi: 10.3389/fnmol.2018.00402

Figure Lengend Snippet: Ultrastructural characterization of PDGFR-β/nestin double-positive cells in the lesion core. Confocal microscopic image of a semi-thin section double-labeled with PDGFR-β and nestin (A,F) , the corresponding transmission electron microscopic image (C,H) , and the overlay image of confocal microscopic data and the corresponding electron microscopic image (B,G) obtained from days 7 (A–E) and 14 (F–K) post-lesion. (D,E,L–K) Higher-magnification views of the boxed areas in (B,C,G,H) , respectively. PDGFR-β/nestin double-positive cells have euchromatic nuclei and rER with dilated cisternae (#). Note that at 7 days, the cytoplasmic processes of these cells are still found extending around the vascular wall on the abluminal side of a smooth muscle (SM) cell and endothelial cells (EN), while at 14 days, they exhibit slender cytoplasmic processes branching out into the extravascular spaces. Cell nuclei are stained with DAPI. Scale bars = 2 μm for (A,B) , and (F–H) ; 1 μm for (C–E,K) ; 0.5 μm for (J) ; 0.2 μm for (I) .

Article Snippet: Primary antibodies and dilutions were as follows: rabbit monoclonal antibody against PDGFR-β (1:200; Abcam), mouse monoclonal antibody against RECA1 (1:200; Bio-Rad, Hercules, CA, USA), chicken polyclonal antibody against glial fibrillary acidic protein (GFAP; 1:500; Millipore), goat polyclonal antibody against type IV collagen (1:100; Bio-Rad), mouse monoclonal antibody against nestin (1:500; Bio-Rad), goat polyclonal antibody against ionized calcium-binding adaptor molecule 1 (Iba1; 1:500; Abcam), chicken polyclonal antibody against vimentin (1:500, Millipore), mouse monoclonal antibody to NG2 (1:500; Millipore), or rabbit polyclonal antibody against Ki-67 (1:1,000; Leica Biosystems, Wetzlar, Germany).

Techniques: Labeling, Transmission Assay, Staining

Journal: Frontiers in Molecular Neuroscience

Article Title: PDGFR-β-Positive Perivascular Adventitial Cells Expressing Nestin Contribute to Fibrotic Scar Formation in the Striatum of 3-NP Intoxicated Rats

doi: 10.3389/fnmol.2018.00402

Figure Lengend Snippet: Schematic representation of dynamic PDGFR-β-positive cells in the lesion core in striata treated with 3-NP. (A) In the saline-treated control striatum, PDGFR-β-positive cells with thin processes are associated with larger caliber vessels having one or two layers of smooth muscle cells. These cells are located outside the smooth muscle cells and are surrounded by the glia limitans of astroglial processes. (B) At 3–7 days post-lesion, PDGFR-β-positive cells are highly proliferative and express nestin and vimentin. They have large euchromatic nuclei and dilated cisternae of rER, indicating the presence of active collagen synthesis. They also migrated to the adjacent microvascular wall and wrap around or directly abut the abluminal surface of endothelial cells or pericytes, despite the ongoing association with the vasculature. (C) At 14–28 days, the lesion core is progressively infiltrated by migrating PDGFR-β-positive cells expressing nestin and vimentin. They have highly branched cytoplasmic processes that are frequently in close apposition or are even interwoven with each other, forming a network. In addition, PDGFR-β-positive somata and processes have close apposition with brain macrophages, nearly always at the branch points of distal processes.

Article Snippet: Primary antibodies and dilutions were as follows: rabbit monoclonal antibody against PDGFR-β (1:200; Abcam), mouse monoclonal antibody against RECA1 (1:200; Bio-Rad, Hercules, CA, USA), chicken polyclonal antibody against glial fibrillary acidic protein (GFAP; 1:500; Millipore), goat polyclonal antibody against type IV collagen (1:100; Bio-Rad), mouse monoclonal antibody against nestin (1:500; Bio-Rad), goat polyclonal antibody against ionized calcium-binding adaptor molecule 1 (Iba1; 1:500; Abcam), chicken polyclonal antibody against vimentin (1:500, Millipore), mouse monoclonal antibody to NG2 (1:500; Millipore), or rabbit polyclonal antibody against Ki-67 (1:1,000; Leica Biosystems, Wetzlar, Germany).

Techniques: Saline, Control, Expressing

Journal: iScience

Article Title: Enhanced differentiation of neural progenitor cells in Alzheimer’s disease into vulnerable immature neurons

doi: 10.1016/j.isci.2025.112446

Figure Lengend Snippet: Enhanced neuronal activity and early differentiation in APPswe cerebral organoids (A–C) Quantification of the perimeter (B) and area (C) of control and APPswe cerebral organoids cultured for one month. n = 80–104, from 3 independent differentiations. Scale bar, 500 μm. (D–F) Neuronal activity was measured by a multi-electrode array in control and APPswe cerebral organoids cultured for one month. n = 11–12, from 3 independent differentiations. (G–I) Immunohistochemistry was performed to analyze the distribution of (H) nesitn- and (I) DCX-positive cells in control and APPswe cerebral organoids cultured for one month. n = 9, from 3 independent differentiations. Scale bar, 20 μm. (J–M) Immunoblotting to determine expression levels of SOX2 (K), DCX (L), and TUJ1 (M) in control and APPswe cerebral organoids cultured for one month. n = 10–14, from 3 independent differentiations. (N–P) Flow cytometry analysis was performed on control and APPswe cerebral organoids cultured for one month to assess the expression levels of (O) nestin- and (P) DCX-positive populations. n = 3 from 3 independent differentiations. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001 (Student’s t test). Error bar ± SEM.

Article Snippet: After blocking, the cells were incubated for 2 hours at 4°C with primary antibodies: a nestin PE-conjugated antibody (R&D Systems) and an antibody against DCX.

Techniques: Activity Assay, Control, Cell Culture, Immunohistochemistry, Western Blot, Expressing, Flow Cytometry

Journal: iScience

Article Title: Enhanced differentiation of neural progenitor cells in Alzheimer’s disease into vulnerable immature neurons

doi: 10.1016/j.isci.2025.112446

Figure Lengend Snippet: Increased mitochondrial ROS and its impact on early differentiation in APPswe NPCs (A–C) Immunocytochemistry analysis of nestin (B) and DCX (C) in control and APPswe NPCs and neurons, differentiated for one week. Scale bar, 40 μm. NPCs, n = 30, from 3 independent differentiations; neuron, n = 75, from 6 independent differentiations. (D) Increased MitoSOX signal in APPswe NPCs. n = 3, from 3 independent differentiations. (E) Reduced utilization of OXPHOS in glucose metabolism in APPswe NPCs. n = 5, from 3 independent differentiations. (F–H) Normalization of increased nestin (G) and decreased DCX (H) expression in APPswe NPCs with EUK8 treatment. Scale bar, 40 μm. n = 75, from 5 independent differentiations. (I) Schematic of the experiment categorizing control NPCs into high-ROS and low-ROS groups based on MitoSOX signal intensity. (J–L) No significant differences in nestin and DCX expression between high-ROS and low-ROS NPC groups. Scale bar, 20 μm. n = 40, from 3 independent differentiations. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001 (ANOVA test followed by Dunnett’s post hoc analysis or Student’s t test). Error bar ± SEM.

Article Snippet: After blocking, the cells were incubated for 2 hours at 4°C with primary antibodies: a nestin PE-conjugated antibody (R&D Systems) and an antibody against DCX.

Techniques: Immunocytochemistry, Control, Expressing

Journal: Cell Death Discovery

Article Title: Cancer stem-like cells can be induced through dedifferentiation under hypoxic conditions in glioma, hepatoma and lung cancer

doi: 10.1038/cddiscovery.2016.105

Figure Lengend Snippet: Hypoxia increased the expression of putative CSC markers. ( a ) In sorted A549 cells, the proportion of CD133-positive cells was only 5.59%±2.272 in the control cells, and this proportion increased in a time-dependent manner under hypoxic conditions. After 15 days of hypoxia, the proportion of CD133-positive cells reached 49.2%±3.125. The CD133 expression in sorted HepG2 cells also increased from 2.04%±1.253 to 40.6%±2.871 after 15 days of hypoxia. For GL261 cells, the proportion of CD133-positive cells was 7.03%±3.425 in the normoxia control cells, and this rate increased remarkably from 9 days to 15 days of hypoxia exposure from 20.3%±2.547 to 97.6%±3.791. In addition, the results also showed that the expression of two other stem cell markers, CD15 and NESTIN, was upregulated, increasing from 0.56%±0.251 to 60.2%±3.472 and 3.96%±5.231 to 60.3%±5.284, respectively, after 15 days of hypoxia. ( b ) The proportion of stem cell markers in sorted cells increased significantly under hypoxic conditions (* P <0.05).

Article Snippet: Sorted cells were exposed to hypoxia for 0, 3, 6, 9, 12 and 15 days, then collected and incubated with an anti-CD133 antibody (Human: Miltenyi Biotech; Mouse: BioLegend, San Diego, CA, USA), anti-CD15 antibody (FAB2155G-100, R&D Systems) and anti-NESTIN antibody (IC2736P, R&D Systems) and analyzed with FCM.

Techniques: Expressing, Control

Journal: Cell Death Discovery

Article Title: Cancer stem-like cells can be induced through dedifferentiation under hypoxic conditions in glioma, hepatoma and lung cancer

doi: 10.1038/cddiscovery.2016.105

Figure Lengend Snippet: The spheres formed by single sorted cancer cells under hypoxia. ( a ) Single cancer cell seeding model; non-cancer stem cells were sorted using magnetic cell sorting, counted and diluted to 1500 cells/1 ml DMEM/F12+10% FBS, and then 1 μ l of the suspension was seeded into each well of 96-well plates containing 170 μ l of DMEM/F12 without serum. ( b , c ) Single sorted (CD133 − cells for A549 and HepG2, CD133 − CD15 − NESTIN − cells for GL261) cancer cells formed a sphere after 21 days of hypoxia exposure; however, the cells under normoxia were dead, and there was no sphere formation (* P <0.05).

Article Snippet: Sorted cells were exposed to hypoxia for 0, 3, 6, 9, 12 and 15 days, then collected and incubated with an anti-CD133 antibody (Human: Miltenyi Biotech; Mouse: BioLegend, San Diego, CA, USA), anti-CD15 antibody (FAB2155G-100, R&D Systems) and anti-NESTIN antibody (IC2736P, R&D Systems) and analyzed with FCM.

Techniques: FACS, Suspension

Journal: Cell Death Discovery

Article Title: Cancer stem-like cells can be induced through dedifferentiation under hypoxic conditions in glioma, hepatoma and lung cancer

doi: 10.1038/cddiscovery.2016.105

Figure Lengend Snippet: Newly formed spheres highly expressed transcription factors and showed asymmetric division. ( a ) Immunofluorescence staining showed that the newly formed GL261 spheres highly expressed SOX-2, OCT-4, KLF-4, Nanog, Lin-28A, CD133, CD15 and NESTIN. ( b ) These newly formed GL261 spheres kept growing in a suspension and proliferated extensively in stem cell culture medium (DMEM/F12+EGF+FGF2+B27) but presented an adherent phenotype in differentiated medium (DMEM/F12+10% FBS).

Article Snippet: Sorted cells were exposed to hypoxia for 0, 3, 6, 9, 12 and 15 days, then collected and incubated with an anti-CD133 antibody (Human: Miltenyi Biotech; Mouse: BioLegend, San Diego, CA, USA), anti-CD15 antibody (FAB2155G-100, R&D Systems) and anti-NESTIN antibody (IC2736P, R&D Systems) and analyzed with FCM.

Techniques: Immunofluorescence, Staining, Suspension, Stem Cell Culture

Journal: Cell Death Discovery

Article Title: Cancer stem-like cells can be induced through dedifferentiation under hypoxic conditions in glioma, hepatoma and lung cancer

doi: 10.1038/cddiscovery.2016.105

Figure Lengend Snippet:

Article Snippet: Sorted cells were exposed to hypoxia for 0, 3, 6, 9, 12 and 15 days, then collected and incubated with an anti-CD133 antibody (Human: Miltenyi Biotech; Mouse: BioLegend, San Diego, CA, USA), anti-CD15 antibody (FAB2155G-100, R&D Systems) and anti-NESTIN antibody (IC2736P, R&D Systems) and analyzed with FCM.

Techniques: