Journal: Neural Regeneration Research

Article Title: Small extracellular vesicles derived from hair follicle neural crest stem cells enhance perineurial cell proliferation and migration via the TGF-β/SMAD/HAS2 pathway

doi: 10.4103/NRR.NRR-D-25-00127

Figure Lengend Snippet: hfNCSC-sEVs enhance tube formation and barrier function in PCs and promote tight junction protein expression. (A) Optical micrographs of the tube formation assay and (B) statistical analyses demonstrated the number of junctions and total length of tubes in PCs in both the phosphate-buffered saline (PBS) and hfNCSC-sEVs groups ( n = 5 per group). (C) Measurements of transmembrane resistance ( n = 3 per group) and (D) cell monolayer permeability assays ( n = 9 per group) indicated the barrier formation ability of PCs in both the PBS and hfNCSC-sEVs groups. (E) Western blot and (F) statistical analyses revealed the relative protein expression levels of the tight junction proteins zonula occludens 1 (ZO1) and claudin-1 in PCs from the PBS and hfNCSC-sEVs groups on day 7 of in vitro culture (normalized to β-actin, n = 3 per group). (G, H) Immunofluorescence staining (G) and statistical analyses (H) showed the integrated optical density (IOD) of ZO1 (green) and the expression of β-tubulin (red) in PCs from the PBS and hfNCSC-sEVs groups on day 7 of in vitro culture ( n = 3 per group). (I) Schematic illustration of the rat sciatic nerve defect model: a 5-mm defect was surgically created in the rat sciatic nerve, which was then bridged using a silicon tube, followed by an orthotopic injection procedure. (J) Immunofluorescence staining revealed the expression of claudin-1 (red) in the proximal end of regenerated tissue in both the PBS and hfNCSC-sEVs groups on day 7 post-operation, with 4′,6-diamidino-2-phenylindole (DAPI) staining indicating the nuclei. Data are expressed as the mean ± SEM. * P < 0.05, *** P < 0.001 (Student’s t -test for B, C, D, F, and H). The data were from at least three separate and independent studies. hfNCSCs: Hair follicle neural crest stem cells; IOD: integrated optical density; PCs: perineurial cells; sEVs: small extracellular vesicles; ZO1: zonula occludens 1.

Article Snippet: The following primary antibodies were used: rabbit polyclonal anti-p75 neurotrophin receptor (p75) antibody (1:100, Cat# 55014-1-AP, Proteintech), mouse monoclonal anti-nestin antibody (1:100, Cat# MAB353, Sigma), rabbit polyclonal anti-claudin-1 antibody (1:250, Cat# 13050-1-AP, Proteintech), rabbit polyclonal anti-ZO1 antibody (1:200, Cat# 21773-1-AP, Proteintech), rabbit polyclonal anti-glucose transporter 1 (GLUT1) antibody (1:500, Cat# 21829-1-AP, Proteintech), rabbit monoclonal anti-S100 antibody (1:800, Cat# MAB353, Abcam), mouse monoclonal anti-neurofilament 200 (NF200) antibody (1:800, Cat# N5389, Sigma), rabbit polyclonal anti-myelin basic protein (MBP) antibody (1:400, Cat# 10458-1-AP, Proteintech), mouse monoclonal anti-β-tubulin antibody (1:1000, Cat# M20005 , Abmart), and rabbit polyclonal anti-HAS2 antibody (1:200, Cat# DF13702, Affinity).

Techniques: Expressing, Tube Formation Assay, Saline, Permeability, Western Blot, In Vitro, Immunofluorescence, Staining, Injection

Journal: Neural Regeneration Research

Article Title: Small extracellular vesicles derived from hair follicle neural crest stem cells enhance perineurial cell proliferation and migration via the TGF-β/SMAD/HAS2 pathway

doi: 10.4103/NRR.NRR-D-25-00127

Figure Lengend Snippet: miR-21-5p in hfNCSC-sEVs augments cell proliferation and migration by enhancing HAS2 expression in PCs. (A, B) Western blot (A) and statistical analyses (B) revealed the relative protein expression levels of HAS2, proliferating cell nuclear antigen (PCNA), and vimentin in PCs across the –/–, –/si- Has2 , hfNCSC-sEVs/–, and hfNCSC-sEVs/si- Has2 groups on day 5 of in vitro culture (normalized to β-actin, n = 3 per group). (C, D) The wound healing assay (C) and statistical analysis (D) demonstrated the migration rates of PCs in the aforementioned groups ( n = 3 per group). (E) The Cell Counting Kit-8 assay was used to assess cell viability in PCs across the same groups on day 5 of in vitro culture ( n = 5 per group). (F, G) Western blot (F) and statistical analyses (G) indicated the relative protein expression levels of HAS2, PCNA, and vimentin in PCs treated with phosphate-buffered saline (PBS), hfNCSC-sEVs, or hfNCSC-sEVs + miR-21-5p inhibitor on day 5 of in vitro culture (normalized to β-actin, n = 3 per group). (H–J) Immunofluorescence staining visualized the expression of HAS2 (red) and 5-ethynyl-2′-deoxyuridine (EdU; green) in PCs (H), and statistical analysis revealed the integrated optical density (IOD) of zonula occludens 1 (ZO1; I) and the cell proliferation rates (J) in the PBS, hfNCSC-sEVs, and hfNCSC-sEVs + miR-21-5p inhibitor groups on day 5 of in vitro culture ( n = 3 per group). (K, L) Western blot (K) and statistical analyses (L) showed the relative protein expression levels of HAS2, PCNA, and vimentin in regenerated tissue from the PBS, hfNCSC-sEVs, and hfNCSC-sEVs + miR-21-5p inhibitor groups on day 5 post-operation (normalized to β-tubulin, n = 3 per group). Data are expressed as the mean ± SEM. ** P < 0.01, *** P < 0.001 (one-way analysis of variance and Tukey’s multiple comparison test for B, D, E, G, I, J, and L). The data were from at least three separate and independent studies. CCK-8: Cell counting kit-8; EdU: 5-ethynyl-2′-deoxyuridine; HAS2: hyaluronan synthase 2; hfNCSCs: hair follicle neural crest stem cells; IOD: integrated optical density; PCNA: proliferating cell nuclear antigen; PCs: perineurial cells; sEVs: small extracellular vesicles; ZO1: zonula occludens 1.

Article Snippet: The following primary antibodies were used: rabbit polyclonal anti-p75 neurotrophin receptor (p75) antibody (1:100, Cat# 55014-1-AP, Proteintech), mouse monoclonal anti-nestin antibody (1:100, Cat# MAB353, Sigma), rabbit polyclonal anti-claudin-1 antibody (1:250, Cat# 13050-1-AP, Proteintech), rabbit polyclonal anti-ZO1 antibody (1:200, Cat# 21773-1-AP, Proteintech), rabbit polyclonal anti-glucose transporter 1 (GLUT1) antibody (1:500, Cat# 21829-1-AP, Proteintech), rabbit monoclonal anti-S100 antibody (1:800, Cat# MAB353, Abcam), mouse monoclonal anti-neurofilament 200 (NF200) antibody (1:800, Cat# N5389, Sigma), rabbit polyclonal anti-myelin basic protein (MBP) antibody (1:400, Cat# 10458-1-AP, Proteintech), mouse monoclonal anti-β-tubulin antibody (1:1000, Cat# M20005 , Abmart), and rabbit polyclonal anti-HAS2 antibody (1:200, Cat# DF13702, Affinity).

Techniques: Migration, Expressing, Western Blot, In Vitro, Wound Healing Assay, Cell Counting, Saline, Immunofluorescence, Staining, Comparison, CCK-8 Assay

Journal: Neural Regeneration Research

Article Title: Small extracellular vesicles derived from hair follicle neural crest stem cells enhance perineurial cell proliferation and migration via the TGF-β/SMAD/HAS2 pathway

doi: 10.4103/NRR.NRR-D-25-00127

Figure Lengend Snippet: miR-21-5p in hfNCSC-sEVs enhances tight junction protein expression in PCs. (A, B) Immunofluorescence staining (A) and statistical analysis (B) demonstrated IOD of ZO1 (green) and the expression of β-tubulin (red) in PCs across the PBS, hfNCSC-sEVs, and hfNCSC-sEVs + miR-21-5p inhibitor groups on day 7 of in vitro culture ( n = 3 per group). (C) Western blot and (D) statistical analyses revealed the relative protein expression levels of the tight junction proteins ZO1 and claudin-1 in PCs from the PBS, hfNCSC-sEVs, and hfNCSC-sEVs + miR-21-5p inhibitor groups on day 7 of in vitro culture (normalized to β-actin, n = 3 per group). (E) Immunofluorescence staining depicted the expression of claudin-1 (red) at the proximal end of regenerated tissue in the PBS, hfNCSC-sEVs, and hfNCSC-sEVs + miR-21-5p inhibitor groups on day 7 post-operation, with DAPI staining highlighting the nuclei. (F, G) Western blot (F) and statistical analyses (G) indicated the relative protein expression levels of ZO1 and claudin-1 in regenerated tissue across the PBS, hfNCSC-sEVs, and hfNCSC-sEVs + miR-21-5p inhibitor groups on day 7 post-operation (normalized to β-actin, n = 3 per group). Data are expressed as the mean ± SEM. ** P < 0.01, *** P < 0.001 (one-way analysis of variance and Tukey’s multiple comparison test for B, D, and G). The data were from at least three separate and independent studies. DAPI: 4,6-Diamidino-2-phenylindole; hfNCSCs: hair follicle neural crest stem cells; IOD: integrated optical density; PBS: phosphate-buffered saline; PCs: perineurial cells; sEVs: small extracellular vesicles; ZO1: zonula occludens 1.

Article Snippet: The following primary antibodies were used: rabbit polyclonal anti-p75 neurotrophin receptor (p75) antibody (1:100, Cat# 55014-1-AP, Proteintech), mouse monoclonal anti-nestin antibody (1:100, Cat# MAB353, Sigma), rabbit polyclonal anti-claudin-1 antibody (1:250, Cat# 13050-1-AP, Proteintech), rabbit polyclonal anti-ZO1 antibody (1:200, Cat# 21773-1-AP, Proteintech), rabbit polyclonal anti-glucose transporter 1 (GLUT1) antibody (1:500, Cat# 21829-1-AP, Proteintech), rabbit monoclonal anti-S100 antibody (1:800, Cat# MAB353, Abcam), mouse monoclonal anti-neurofilament 200 (NF200) antibody (1:800, Cat# N5389, Sigma), rabbit polyclonal anti-myelin basic protein (MBP) antibody (1:400, Cat# 10458-1-AP, Proteintech), mouse monoclonal anti-β-tubulin antibody (1:1000, Cat# M20005 , Abmart), and rabbit polyclonal anti-HAS2 antibody (1:200, Cat# DF13702, Affinity).

Techniques: Expressing, Immunofluorescence, Staining, In Vitro, Western Blot, Comparison, Saline

Journal: bioRxiv

Article Title: Scp160 deletion suppresses TDP-43 aggregation and toxicity in Saccharomyces cerevisiae

doi: 10.64898/2026.04.28.721091

Figure Lengend Snippet: (A) Ribosome sedimentation profiles of WT, scp160Δ , and bfr1Δ cells. Cells were grown in YPDA to mid-log phase and lysed under polysome-preserving conditions. Lysates were centrifuged through 15-45% sucrose gradients and fractionated with the continuous measurement of absorbance at 256 nm to visualize ribosomal species peaks. Positions of 40S and 60S ribosomal subunits, 80S monosomes, and polysomes are indicated. (B) Semi-quantitative immunoblot analysis of TDP43-GFP expression in WT, bfr1Δ , and scp160Δ cells carrying P GAL1 -TDP43-GFP. Cells were grown on raffinose and then shifted to either glucose-repressing conditions or to galactose for induction for 4 h. TDP43-GFP was detected with an anti-GFP antibody; tubulin was used as a loading control. (C) Immunoblot analysis of secretory-protein reporter Rny1-FLAG in WT, scp160Δ , and bfr1Δ cells. Rny1-FLAG (wild-type (wt) or the glycosylation-defective Rny1-FLAG W399R mutant) was expressed from the 2 m plasmid from the ADH promoter. Rny1-FLAG species were detected with anti-FLAG antibody. Tubulin was used as a loading control.

Article Snippet: Monoclonal anti-GFP antibodies were from Santa Cruz Biotechnology (sc-9996) and used at 800 ng/mL; mouse monoclonal anti-α-tubulin antibodies (12G10) were obtained from the Developmental Studies Hybridoma Bank (DSHB; University of Iowa, Iowa City, IA, USA) and used at a 1:1000 dilution; mouse monoclonal anti-FLAG antibodies (M2) were purchased from Sigma and used at 1 μg/mL; anti-mouse IgG-HRP were from GE-Healthcare (NA931) and used at a dilution of 1:5000.

Techniques: Sedimentation, Preserving, Western Blot, Expressing, Control, Glycoproteomics, Mutagenesis, Plasmid Preparation

Journal: PLOS One

Article Title: Differential regulation of p62-ubiquitin conjugates in neurons versus astrocytes during cellular stress

doi: 10.1371/journal.pone.0345890

Figure Lengend Snippet: (A) Schematic of the neuron-astrocyte coculture . (B) Representative images of the neuron-astrocyte coculture. Coculture of GFP-LC3 transgenic astrocytes and non-transgenic neurons were fixed on DIV7 of coculture (neurons were a total of DIV11) and immunostained for GFP (LC3; labels GFP-LC3 transgenic astrocytes), AQP4 (astrocyte marker) and β3-tubulin (neuron marker); nuclei were labeled with Hoechst (shown in blue in the merged image). Shown are maximum projections of z-stacks. Dashed box indicates location of the zoom-in. Scale bars, 10 µm. (C) Cocultured neurons and astrocytes were treated for 1 hr with 1 mM LLOMe to induce lysosomal damage or an equivalent volume of DMSO solvent as a control. Cocultures were immunostained for GFP (LC3; labels GFP-LC3 transgenic astrocytes), p62, and LAMP1. Shown are maximum projections of z-stacks. Individual images are shown in grayscale; images for GFP(LC3), p62, and LAMP1 are grayscale-matched within a protein marker, across treatment conditions. Filled yellow arrowheads indicate puncta co-positive for GFP(LC3) and LAMP1, or p62 and LAMP1. Empty yellow arrowheads indicate GFP(LC3) or p62 puncta that are negative for LAMP1. Scale bars, 10 µm. ( C’ ) Representative line scans of p62 and LAMP1-positive puncta in the soma of neurons and astrocytes. Asterisks denote peaks co-positive for p62 and LAMP1. (D) Cocultured neurons and astrocytes were treated for 1 hr with 1 mM LLOMe to induce lysosomal damage or an equivalent volume of DMSO solvent as a control. Cocultures were immunostained for GFP (LC3; labels GFP-LC3 transgenic astrocytes), p62, and ubiquitin; nuclei (Nuc) were labeled with Hoechst. Shown are maximum projections of z-stacks. Individual images are shown in grayscale; images for p62 and ubiquitin are grayscale-matched within a protein marker, across treatment conditions. Filled yellow arrowheads indicate colocalization between p62 puncta and ubiquitin puncta. Empty yellow arrowheads indicate p62 puncta with no ubiquitin puncta correlate. Scale bar, 10 µm. (E-E’) Quantification of the total area occupied by p62-positive puncta normalized to soma area for neurons (E) or astrocytes (E’) . Horizontal bars represent the means of the biological replicates ± SEM; shown are p-values from a LME model; N = 37-40 neurons and N = 27-33 astrocytes from 3 independent experiments; 7-8 DIV of coculture. (F-F’) Quantification of the total area occupied by ubiquitin-positive puncta normalized to soma area for neurons (F) or astrocytes (F’) . Horizontal bars represent the means of the biological replicates ± SEM; shown are p-values from a LME model; N = 35-36 neurons and N = 29-33 astrocytes from 3 independent experiments; 7-8 DIV of coculture. (G-G’) Quantification of the percentage of overlapping area between p62-positive puncta and ubiquitin-positive puncta normalized to soma area for neurons (G) or astrocytes (G’) . Horizontal bars represent the means of the biological replicates ± SEM; shown are p-values from a LME model; N = 32-33 neurons and N = 23-30 astrocytes from 3 independent experiments; 7-8 DIV of coculture. Throughout the figure, neurons are circled in orange and astrocytes are circled in purple. For all graphs in all figures, small circles indicate the measurements from individual cells (e.g., the technical replicates) from each of the independent experiments; large triangles indicate the corresponding biological means from each of the independent experiments (e.g., the biological replicates); independent experiments are color-coded.

Article Snippet: Primary antibodies for immunofluorescence include mouse anti-βIII Tubulin (R&D Systems, MAB1195), rabbit anti-AQP4 (Millipore Sigma, HPA014784), chicken anti-GFP (Aves Labs, Inc., GFP-1020), rabbit anti-p62 (Abcam, Ab109012 ), mouse anti-Ubiquitin (Enzo Life Sciences, ENZ-ABS840; − and ), mouse anti-Ubiquitin (Enzo Life Sciences, BML-PW8810; , and ), and rat anti-LAMP1 (Abcam, ab25245).

Techniques: Transgenic Assay, Marker, Labeling, Solvent, Control, Ubiquitin Proteomics