Journal: Non-coding RNA Research

Article Title: Exosomal miRNA-218–5p derived from low-passage dermal papilla cells modulates hair follicle growth and development

doi: 10.1016/j.ncrna.2026.01.004

Figure Lengend Snippet: MiR-218-5p regulated HF growth- and development-related gene expression in HFSCs. (A) MiR-218–5p expression levels in HFSCs after transfection with miR-218–5p mimics or the inhibitor (unpaired two-tailed t -test, n = 3). (B) Expression of HF development-related genes in HFSCs is regulated by miR-218–5p. (C) β-Catenin and SFRP2 protein expression in HFSCs after treatment with miR-218–5p mimics or inhibitor (unpaired two-tailed t -test, n = 3). ∗ P < 0.05, ∗∗ P < 0.01.

Article Snippet: Anti-SFRP2 rabbit polyclonal antibody (Proteintech Biotech, Cat No. 12189-1-AP), anti-β-catenin polyclonal antibody (Proteintech, China, Cat No. 51067-2-AP), and anti-GAPDH mouse monoclonal antibody (Proteintech, China, Cat No. 60004-1-Ig) were the primary antibodies.

Techniques: Gene Expression, Expressing, Transfection, Two Tailed Test

Journal: Non-coding RNA Research

Article Title: Exosomal miRNA-218–5p derived from low-passage dermal papilla cells modulates hair follicle growth and development

doi: 10.1016/j.ncrna.2026.01.004

Figure Lengend Snippet: A schematic showing exosomal miR-218–5p derived from DPCs positively regulating HF growth by targeting SFRP2.

Article Snippet: Anti-SFRP2 rabbit polyclonal antibody (Proteintech Biotech, Cat No. 12189-1-AP), anti-β-catenin polyclonal antibody (Proteintech, China, Cat No. 51067-2-AP), and anti-GAPDH mouse monoclonal antibody (Proteintech, China, Cat No. 60004-1-Ig) were the primary antibodies.

Techniques: Derivative 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 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: 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 monoclonal anti-proliferating cell nuclear antigen (PCNA) antibody (1:2000, Cat# 60097-1-Ig, Proteintech, Wuhan, China), rabbit monoclonal anti-vimentin antibody (1:1000, Cat# 5741, Cell Signaling Technology, Danvers, MA, USA), rabbit polyclonal anti-claudin-1 antibody (1:1000, Cat# 13050-1-AP, Proteintech), rabbit polyclonal anti-zonula occludens 1 (ZO1) antibody (1:10 000, Cat# 21773-1-AP, Proteintech), rabbit polyclonal anti-mothers against decapentaplegic homolog (SMAD)7 antibody (1:500, Cat# WL02975, Wanleibio, Shenyang, China), rabbit polyclonal anti-SMAD2/3 antibody (1:1000, Cat# WL01520, Wanleibio), rabbit polyclonal anti-p-SMAD2/3 antibody (1:500, Cat# WL02305, Wanleibio), rabbit recombinant anti-hyaluronan synthase 2 (HAS2) antibody (1:500, Cat# DF13702, Affinity, Cincinnati, OH, USA), rabbit monoclonal anti-β-actin antibody (1:1000, Cat# 4970, Cell Signaling Technology), and mouse monoclonal anti-β-tubulin antibody (1:5000, Cat# M20005 , Abmart, Shanghai, China).

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: 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 monoclonal anti-proliferating cell nuclear antigen (PCNA) antibody (1:2000, Cat# 60097-1-Ig, Proteintech, Wuhan, China), rabbit monoclonal anti-vimentin antibody (1:1000, Cat# 5741, Cell Signaling Technology, Danvers, MA, USA), rabbit polyclonal anti-claudin-1 antibody (1:1000, Cat# 13050-1-AP, Proteintech), rabbit polyclonal anti-zonula occludens 1 (ZO1) antibody (1:10 000, Cat# 21773-1-AP, Proteintech), rabbit polyclonal anti-mothers against decapentaplegic homolog (SMAD)7 antibody (1:500, Cat# WL02975, Wanleibio, Shenyang, China), rabbit polyclonal anti-SMAD2/3 antibody (1:1000, Cat# WL01520, Wanleibio), rabbit polyclonal anti-p-SMAD2/3 antibody (1:500, Cat# WL02305, Wanleibio), rabbit recombinant anti-hyaluronan synthase 2 (HAS2) antibody (1:500, Cat# DF13702, Affinity, Cincinnati, OH, USA), rabbit monoclonal anti-β-actin antibody (1:1000, Cat# 4970, Cell Signaling Technology), and mouse monoclonal anti-β-tubulin antibody (1:5000, Cat# M20005 , Abmart, Shanghai, China).

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

Journal: Oncology Letters

Article Title: RNA methyltransferase NSUN2 enhances vasculogenic mimicry and malignant progression of cervical cancer through upregulation of MMP-9

doi: 10.3892/ol.2026.15518

Figure Lengend Snippet: MMP-9 , a factor that promotes Vasculogenic mimicry, is highly expressed in CC and is associated with poor prognosis. (A) CC database of TCGA was used to analyze key factors associated with VM. (B) Association of Sox2 expression with overall survival in CC (log-rank test). (C) Association of MMP-9 expression with overall survival in CC (log-rank test). (D) Panoramic scans after immunohistochemical detection of MMP-9 and H&E staining in samples from cancerous and paracancerous tissues from subjects with CC. Scale bar, 50 µm. Original magnification, ×20. (E) Protein levels of MMP-9 in 20 paired samples, with the MMP-9 level in CC tissue expressed compared with that in the paired normal tissue. (F) Expression levels of MMP-9 mRNA in 44 paired CC and paracancerous tissues, with MMP-9 expression in CC tissue expressed compared with that in the paired normal tissue. (G) Comparison of the average expression levels of MMP-9 mRNA in CC tissues compared with paracancerous tissues. (H) HeLa and SiHa cells were incubated under hypoxia (0.1% O 2 ) and proteins collected at 24, 48 and 72 h for western blotting of ALDH1, EPHA2, MMP-9 and GAPDH. ImageJ was used to semi-quantify western blotting signals from HeLa (I) and SiHa (J) cells. GAPDH served as an internal reference. *P<0.05, **P<0.01 and ***P<0.001. MMP-9, matrix metalloproteinase 9; VM, vasculogenic mimicry; ALDH1, aldehyde dehydrogenase 1; EPHA2, ephrin type-A receptor 2; TCGA, The Cancer Genome Atlas; Sox2, SRY-box transcription factor 2; CC, cervical cancer; CESC, cervical squamous cell carcinoma.

Article Snippet: The membranes were blocked with 5% milk at 20±5°C for 1 h. The blocked membranes were incubated at 4°C overnight with the following antibodies: A rabbit monoclonal anti-NSUN2 antibody (1:1,000; cat. no. AB259941; Abcam), a rabbit monoclonal anti-transfer RNA aspartic acid methyltransferase 1 (TRDMT1) antibody (1:1,000; cat. no. 19221-1-AP; Proteintech Group, Inc.; Wuhan Sanying Biotechnology), a rabbit polyclonal anti-MMP-9 antibody (1:1,000; cat. no. 10375-2-AP; Proteintech Group, Inc.; Wuhan Sanying Biotechnology), a rabbit polyclonal anti-aldehyde dehydrogenase 1 (ALDH1) antibody (1:1,000; cat. no. 15910-1-AP; Proteintech Group, Inc.; Wuhan Sanying Biotechnology), a rabbit polyclonal anti-ephrin type-A receptor 2 (EPHA2) antibody (1:1,000; cat. no. AF5 238; Affinity Biosciences) and a rabbit polyclonal anti-GAPDH antibody (1:1,000; TA309157 OriGene Technologies, Inc.).

Techniques: Expressing, Immunohistochemical staining, Staining, Comparison, Incubation, Western Blot

Journal: iScience

Article Title: Stimulation to secrete insulin induces pancreatic β-cell dysfunction through Tfe3 activation

doi: 10.1016/j.isci.2026.115312

Figure Lengend Snippet: Tfe3 is activated by stimuli to secrete insulin (A) Schematic representation of the long and short isoforms of Tfe3. Previously identified phosphoracceptor Ser/Thr residues are indicated. Rag-BD, Rag-binding domain; TAD, transactivation domain; bHLH-zip, basic-helix-loop-helix zipper domain. (B) Glucose stimulation induces de-phosphorylation of Tfe3. Extracts of MIN6 cells grown in the presence of 12.5 mM glucose or stimulated with 25 mM glucose for 6 h were analyzed by immunoblotting using an anti-Tfe3 antibody ( n = 3). Two Tfe3 isoforms and their phosphorylated and non-phosphorylated species are indicated. Representative blots of three independent experiments are shown. (C) KCl stimulation induces de-phosphorylation of Tfe3. Extracts of MIN6 cells treated with KCl for 1 h were analyzed by immunoblotting using an anti-Tfe3 antibody ( n = 3). (D) KCl stimulation of MIN6 cells induces nuclear accumulation of Tfe3. Cells treated with KCl (20 mM) and/or nimodipine (10 μM) were stained with an anti-Tfe3 antibody (top) and DAPI (bottom). Quantification of nuclear Tfe3 signal intensities in a representative experiment ( n = 2) is shown in a boxplots (right). In total, >788 cells were counted per condition per repeat. Scale bar, 10 μm. (E) KCl stimulation induces nuclear accumulation of Tfe3 in primary islet cells. Primary islets were treated with KCl (40 mM) and/or nimodipine (10 μM). Whole islets were stained with an anti-Tfe3 antibody (middle) and DAPI (bottom) and imaged using a confocal microscope. Top, shows merged images of Tfe3 and DAPI. Representative images from four independent experiments are shown. Scale bar, 20 μm. (F) Immunofluorescence staining of Tfe3 (red) in pancreas sections from a pair of mice (pair #1, identical to that shown in F) given water or 20% glucose-supplemented water for 18 days. The sections were also stained for insulin (green) together with nuclear counterstaining with DAPI (blue). Representative images of sections from the pair are shown ( n = 3). Sections from each pair were simultaneously processed for staining and images were acquired under identical settings. Nuclear Tfe3 signals in the pair of mice were quantified and are presented in violin and boxplots on the right. Cell numbers counted in each condition are also shown. Examination of two other pairs of samples yielded similar results ( C). Scale bar, 50 μm. ∗∗∗ p < 0.001; Kruskal-Wallis test followed by Steel-Dwass test (D) or Mann-Whitney U test (F). Boxes in (D) show median and lower and upper quantiles, and whiskers show 1.5 × interquartile range (IQR). The cross marks denote the averages. See also .

Article Snippet: Rabbit polyclonal anti-TFE3 (for ChIP and IF) , Proteintech , Cat# 14480-1-AP; RRID: AB_2199587.

Techniques: Binding Assay, De-Phosphorylation Assay, Western Blot, Staining, Microscopy, Immunofluorescence, MANN-WHITNEY

Journal: iScience

Article Title: Stimulation to secrete insulin induces pancreatic β-cell dysfunction through Tfe3 activation

doi: 10.1016/j.isci.2026.115312

Figure Lengend Snippet: KCl stimulation downregulates Mafa by activating Tfe3 (A–C) Enforced expression of Tfe3 suppresses endogenous Mafa levels. (A) Schematic structure of the expression plasmids and the Δ N1 mutant of Tfe3. EF1α, elongation factor 1α promoter; CMV, cytomegalovirus immediate-early enhancer and promoter. (B) MIN6 cells were transfected with the indicated plasmid and stained with anti-EGFP (green, on left) and anti-Mafa (red, on right) antibodies. Nuclei were counterstained with DAPI (blue). Arrows indicate EGFP-positive cells that received the plasmid. Mafa signal intensities in EGFP-negative and EGFP-positive cells in each condition in a representative experiment ( n = 3) were quantified and are presented in beeswarm and boxplots. Cell numbers counted are shown in the plots. Scale bar, 5 μm. (C) MIN6 cells were transfected with EGFP-Tfe3 Δ N1 expression plasmid and stained with anti-EGFP (green) and anti-Tfe3 (red) antibodies. Nuclei were counterstained with DAPI (blue). The rightmost panel shows a longer exposure image of the middle. Arrows and asterisks indicate EGFP-positive and EGFP-negative cells that received or did not receive the plasmid, respectively. Scale bar, 10 μm. (D–F) Knockdown of Tfe3 blunts KCl-stimulated downregulation of Mafa. (D) Schematic structure of the shRNA expression plasmids. (E) MIN6 cells transfected with the indicated plasmid were treated with 20 mM KCl for 6 h and subjected to immunofluorescence staining using anti-Tfe3 and anti-EGFP antibodies. The endogenous Tfe3 signal intensities were quantified and are presented in beeswarm and boxplots ( n = 2). Cell numbers counted in each condition are shown. (F) MIN6 cells transfected with the indicated plasmid were treated with 20 mM KCl for 6 h and subjected to immunofluorescence staining using anti-Mafa (red, top) and anti-EGFP (green, bottom) antibodies. Nuclei were stained with DAPI (blue). Arrows indicate EGFP-positive cells that were successfully transfected with the plasmids. Nuclear Mafa signal intensities in EGFP-negative and EGFP-positive cells were separately quantified and are presented in beeswarm and boxplots (right). Quantification of a representative experiment ( n = 2) and cell numbers counted in each condition are shown. Scale bar, 10 μm. ∗ p < 0.05; ∗∗∗ p < 0.001; n.s., not significant; Kruskal-Wallis test followed by Steel-Dwass test (B, E, and F).

Article Snippet: Rabbit polyclonal anti-TFE3 (for ChIP and IF) , Proteintech , Cat# 14480-1-AP; RRID: AB_2199587.

Techniques: Expressing, Mutagenesis, Transfection, Plasmid Preparation, Staining, Knockdown, shRNA, Immunofluorescence

Journal: iScience

Article Title: Stimulation to secrete insulin induces pancreatic β-cell dysfunction through Tfe3 activation

doi: 10.1016/j.isci.2026.115312

Figure Lengend Snippet: Organelle stress and starvation activate Tfe3 and downregulate Mafa (A) Schematic view of the insulin biosynthesis and secretory pathway and sites of action of the organelle stressors used. (B) Tfe3 nuclear accumulation induced by stress-inducing reagents. MIN6 cells were treated with the indicated compounds (Cq, 20 mM; BfA, 20 μM; Mo, 50 nM; Tp, 100 nM; and Tu, 1 μM) for 6 h and stained with an anti-Tfe3 antibody (red, top) and DAPI (blue, bottom). Signal intensities of nuclear Tfe3 in a representative experiment ( n = 2) were quantified and are summarized in a boxplots (right). In total, >620 cells were counted per condition per repeat. Scale bar, 10 μm. (C) Organelle stresses reduce Mafa and Neurod1 levels in MIN6 insulinoma cells. Cells were treated with the indicated stressors for 6 h, and the cell extracts were analyzed by immunoblotting using the indicated antibodies. Quantification of the blot relative to βActin levels is indicated on the right (mean ± SEM; n = 3). (D) MIN6 cells grown in D-MEM supplemented with 12.5 mM glucose and 15% fetal bovine serum (Ctrl) were starved (0 mM glucose and 0% fetal bovine serum) for 24 h and stained with an anti-Tfe3 antibody (red, top). Tfe3 signal intensities in nuclei (blue) in a representative experiment ( n = 2) were quantified and are presented in a boxplot. In total, >567 cells were counted per condition per repeat. Scale bar, 20 μm. (E) Extracts from growing (Ctrl) or starved (Stv) MIN6 cells as in (D) were blotted with an anti-Mafa antibody. Quantification of the blot relative to βActin levels is indicated on the right (mean ± SEM; n = 3). (F) The mRNA levels of Mafa and Gapdh in MIN6 cells as in (D) were analyzed by quantitative RT-PCR (mean ± SEM; n = 3). (G and H) Immunostaining of pancreas sections from mice fed al libitum (Ctrl) or fasted for 16 h (Fasting) with the indicated antibodies. Images are representative of two independent experiments. Sections from each pair of mice were simultaneously processed for staining and images were acquired under identical settings. Quantification of nuclear Tfe3 (G) and Mafa (H) intensities in a pair of mice is indicated in violin plots on the right. Cell numbers counted are shown in the plots. Examination of another pairs of samples yielded similar results ( C and S5D). Scale bars, 50 μm. ∗ p < 0.05; ∗∗∗ p < 0.001; n.s., not significant; one-way ANOVA followed by Tukey’s multiple comparison test (B and C), Mann-Whitney U test (D, G, and H), or Student’s t test (E and F). See also .

Article Snippet: Rabbit polyclonal anti-TFE3 (for ChIP and IF) , Proteintech , Cat# 14480-1-AP; RRID: AB_2199587.

Techniques: Staining, Western Blot, Quantitative RT-PCR, Immunostaining, Comparison, MANN-WHITNEY