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 are taken up by PCs in vitro and enhance their proliferation and migration. (A) Primary cultures of hfNCSCs were established from male Sprague–Dawley rats. (B) Immunofluorescence staining of the neural crest cell marker p75 (red) and the stem cell marker nestin (green) in hfNCSCs, with 4′,6-diamidino-2-phenylindole (DAPI) staining indicating the nuclei. (C) Western blot analysis demonstrated the presence of surface markers (cluster of differentiation [CD]9, CD81, and tumor susceptibility gene 101 protein [TSG101]) and the absence of an endoplasmic reticulum marker (calnexin) in hfNCSC-sEVs. (D) Nanoparticle tracking analysis was used to quantify the concentration and size distribution of hfNCSC-sEVs. (E) Transmission electron microscopy was used to visualize the characteristic morphology of hfNCSC-sEVs. (F) Immunofluorescence staining indicated that the third-generation PCs cultured in vitro were positive for claudin-1, zonula occludens 1 (ZO1), and glucose transporter 1 (GLUT1) but negative for S100, with DAPI staining marking the nuclei. (G) The internalization of PKH26-labeled hfNCSC-sEVs (red) by ZO1-positive PCs (green) was visualized using immunofluorescence staining, with DAPI staining to mark the nuclei. (H) The Cell Counting Kit-8 assay was used to evaluate the cell viability of PCs across concentrations of 0, 2 × 10 8 , 5 × 10 8 , and 10 × 10 8 particles/mL hfNCSC-sEVs at 3, 5, and 7 days of in vitro culture ( n = 5 per group). (I) The Transwell assay was used to quantify the number of migrating PCs at 6, 12, and 18 hours post-treatment with the aforementioned concentrations of hfNCSC-sEVs, in in vitro culture ( n = 6 per group). (J) Western blot and (K) statistical analyses revealed the relative protein expression levels of proliferating cell nuclear antigen (PCNA) and vimentin in PCs from the phosphate-buffered saline (PBS) and hfNCSC-sEVs groups on day 5 of in vitro culture (normalized to β-actin, n = 3 per group). Data are expressed as the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 (one-way analysis of variance and Tukey’s multiple comparison test for H and I; Student’s t -test for K). The data were from at least three separate and independent studies. CCK-8: Cell counting kit-8; GLUT1: glucose transporter 1; hfNCSCs: hair follicle neural crest stem cells; ns: not significant; 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: In Vitro, Migration, Immunofluorescence, Staining, Marker, Western Blot, Concentration Assay, Transmission Assay, Electron Microscopy, Cell Culture, Labeling, Cell Counting, Transwell Assay, Expressing, Saline, 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: 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: Bioactive Materials

Article Title: A promising magnesium-related alloy with metabolic reprogramming and antitumor effects in hepatocellular and pancreatic cancer

doi: 10.1016/j.bioactmat.2025.12.039

Figure Lengend Snippet: Transcriptomic Analysis Indicates Mg and Al-Mg Alter Gene Expression Patterns in Hepatocellular and Pancreatic Cancer Cells. (A) High-throughput sequencing of PANC-1, PANC-1-Mg, PANC-1-Al-Mg, Huh7, Huh7-Mg, and Huh7-Al-Mg groups; heatmap showing sample correlations. (B) Volcano plots illustrating differential gene expression in Huh7 and PANC-1 cells after Mg or Al-Mg treatment. (C) GO enrichment analysis of differentially expressed genes following Mg or Al-Mg treatment. (D) KEGG pathway enrichment analysis of differentially expressed genes after Mg or Al-Mg treatment. (E) GSEA enrichment analysis of gene expression profiles post Mg or Al-Mg treatment. (F) Western blot analysis of AMPK, p-AMPK and CPT1B expression in PANC-1 cells after Mg or Al-Mg exposure with or without AMPK agonist. (G) PPI network analysis of differentially expressed genes in Mg or Al-Mg-treated cells. (H) Integrated analysis of metabolomic and transcriptomic data by MetaboAnalyst 6.0.

Article Snippet: After blocking with 5 % nonfat milk for 1 h at room temperature, membranes were incubated overnight at 4 °C with primary antibodies, including AMPK (1:1000, CST, 2532), p-AMPK (1:1000, CST, 2535), CPT1B (1:1000, Proteintech, 22170-1-AP), CDK4 (1:1000, Proteintech, 11026-1-AP), PCNA (1:1000, Proteintech, 10205-2-AP), p21 (1:1000, Proteintech, 10355-1-AP), GAPDH (1:1000, Proteintech, 60004-1-Ig) followed by HRP-conjugated secondary antibody (1:5000, Proteintech, RGAR001) for 1 h at room temperature.

Techniques: Gene Expression, Next-Generation Sequencing, Western Blot, Expressing

Journal: Bioactive Materials

Article Title: A promising magnesium-related alloy with metabolic reprogramming and antitumor effects in hepatocellular and pancreatic cancer

doi: 10.1016/j.bioactmat.2025.12.039

Figure Lengend Snippet: Metabolomic Analysis Reveals Mg and Al-Mg Induce Metabolic Reprogramming in Hepatocellular and Pancreatic Cancer Cells. (A) Metabolomic profiling of PANC-1, PANC-1-Mg, PANC-1-Al-Mg, Huh7, Huh7-Mg, and Huh7-Al-Mg groups using LC-MS identified 1824 metabolites. (B) PCA illustrating clustering among different cell groups. (C) Heatmap showing differential abundances of characteristic metabolites across cell groups. (D) K-means clustering analysis highlighting metabolic differences among the cell groups. (E) Venn diagram displaying common differential metabolites among treatment groups. (F) Volcano plots of differential metabolites following Mg or Al-Mg treatment. (G) KEGG pathway enrichment analysis of differential metabolites. (H) Enrichment distribution of differential metabolites in Huh7 or PANC-1 cells treated with Mg or Al-Mg. (I) Quantitative analysis of intracellular metabolites including L-glutamine, adenine, uridine, cytidine, and guanine by ELISA with Mg or Al-Mg exposure. (J) Western blot analysis of p21, CDK4, and PCNA expression in PANC-1 cells after Mg or Al-Mg exposure. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: After blocking with 5 % nonfat milk for 1 h at room temperature, membranes were incubated overnight at 4 °C with primary antibodies, including AMPK (1:1000, CST, 2532), p-AMPK (1:1000, CST, 2535), CPT1B (1:1000, Proteintech, 22170-1-AP), CDK4 (1:1000, Proteintech, 11026-1-AP), PCNA (1:1000, Proteintech, 10205-2-AP), p21 (1:1000, Proteintech, 10355-1-AP), GAPDH (1:1000, Proteintech, 60004-1-Ig) followed by HRP-conjugated secondary antibody (1:5000, Proteintech, RGAR001) for 1 h at room temperature.

Techniques: Liquid Chromatography with Mass Spectroscopy, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing