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: Science Advances

Article Title: Engineered anchoring aptamers induce relocalization and functional inactivation of transcription factors

doi: 10.1126/sciadv.adw6496

Figure Lengend Snippet: ( A ) Schematic illustration of cytoplasmic relocalization of p65 mediated by the anchoring aptamer. ( B ) Schematic diagram of the p65 ligand (P028F4, an aptamer targeting p65), the Ras ligand (Ra1, an aptamer targeting Ras) and anchoring aptamers [PAR (P028F4-AAA-Ra1), RAP (Ra1-AAA-P028F4), PTR (P028F4-TTT-Ra1), and RTP (Ra1-TTT-P028F4)]. ( C ) Native polyacrylamide gel analysis of aptamers. Lane 1, single-stranded DNA (ssDNA) marker; lane 2, P028F4; lane 3, Ra1; lane 4, PAR; lane 5, RAP; lane 6, PTR; lane 7, RTP. nt, nucleotides. ( D ) H1299 cells were transfected with control library, PAR, RAP, PTR, or RTP (all at 200 nM) for 12 hours, followed by tumor necrosis factor–α (TNFα) stimulation (10 ng/ml for 1.5 hours). Cytoplasmic (cyto) and nuclear (nuc) p65 levels were analyzed by cellular fractionation and immunoblotting. Error bars represent means ± SD from three independent experiments ( n = 3). P values are indicated by Student’s t test. ( E ) H1299 cells were transfected with 200 nM RTP constructs containing linkers of different lengths for 12 hours, followed by TNFα stimulation (10 ng/ml for 1.5 hours). Cytoplasmic and nuclear p65 levels were analyzed by cellular fractionation and immunoblotting. ( F ) H1299 cells were transfected with RTP at varying concentrations (0, 100, 200, and 300 nM) for 12 hours, followed by TNFα stimulation (10 ng/ml for 1.5 hours). Cytoplasmic and nuclear p65 levels were assessed by cellular fractionation and immunoblotting. Proliferating cell nuclear antigen (PCNA) or H3 serves as the nuclear marker, and β-actin or vinculin serves as the cytoplasmic marker.

Article Snippet: The anti-Ras antibody (18295-1-AP), anti-E2F1 antibody (66515-1-Ig), anti-vinculin antibody (66305-2-Ig), anti-H3 antibody (68345-1-Ig), and anti-proliferating cell nuclear antigen (PCNA) antibody (60097-1-Ig) were obtained from Proteintech.

Techniques: Marker, Transfection, Control, Cell Fractionation, Western Blot, Construct

Journal: Biomedical Reports

Article Title: TiO 2 NPs improve ultrasound response: CS/β-GP/TiO 2 NP hydrogel enabling on-demand administration

doi: 10.3892/br.2025.2020

Figure Lengend Snippet: Western blotting results of different composite hydrogels. The results of western blotting assay revealed no significant differences in cell protein expression among the different groups (P>0.05). PCNA, proliferating cell nuclear antigen; CS/β-GP, chitosan/β-glycerophosphate; TiO 2 NPs, titanium dioxide nanoparticles.

Article Snippet: The following materials were used: i) CS powder (95% deacetylation degree; cat. no. C105799; Aladdin Scientific Corp.), ii) β-GP pentahydrate (cat. no. D106347; Aladdin Scientific Corp.), iii) sodium fluorescein (NaF; cat. no. F105615; Aladdin), iv) TiO 2 NPs (20-40 nm; cat. no. NM000800; Beijing Solarbio Science & Technology Co., Ltd.), v) L929 murine fibroblast cells (cat. no. KGG1306-1), vi) RPMI-1640 medium (containing newborn calf serum, double antibiotics; cat. no. KGL1509-500), vii) Cell Counting Kit-8 (CCK-8) cell proliferation assay kit (cat. no. KGA9305-500), viii) LIVE/DEAD cell viability assay kit (cat. no. KGA9501-1000), ix) bovine serum albumin (BSA) (standard grade, heat-treated) (cat. no. KGL2314-10), x) bicinchoninic acid (BCA) protein quantification assay kit (cat. no. KGB2101-250), all from Jiangsu KeyGen Biotech Co., Ltd., xi) mouse anti-proliferating cell nuclear antigen (PCNA) antibody (1:1,000; cat. no. BM0104) and xii) goat anti-mouse IgG/HRP antibody (1:10,000; cat. no. BA1056) both from Boster Biological Technology Co. Ltd.

Techniques: Western Blot, Expressing, Titanium Dioxide

Journal: PLOS One

Article Title: Cysteine dioxygenase knockout and taurine deficiency impair mouse uterine adenogenesis by inhibiting epithelial cell proliferation and enhancing apoptosis

doi: 10.1371/journal.pone.0329503

Figure Lengend Snippet: (a-d) 10 days taurine supplementation to the Cdo KO mice (KO+Tau) increases the taurine level (a), the number of uterine glands marked by Foxa2 (b, c) and Foxa2 mRNA levels (d) in the uterine tissue. (e) Western Blot detection and analysis of PCNA protein levels in the uteri of the WT, Cdo KO and KO + Tau mice (n ≥ 3). (f) Western blot detection of BAX/BCL2 protein levels (up) and the analysis of BAX/BCL2 ration (down) in the uteri of the WT, Cdo KO and KO + Tau mice. WT: wild type. KO: Cdo KO. PND: Postnatal day. Scale bar represents 20 μm. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: The membrane was blocked with 5% (w/v) nonfat dry milk in 0.05 mol/L pH 7.4 Tris buffered saline (TBS) for 1 h and incubated with rabbit anti-CDO antibody (ab53436, abcam, Cambridge, UK; 1:2000), mouse anti- proliferating cell nuclear antigen (PCNA) antibody (60097–1-Ig, Proteintech Group, Inc., IL, USA; 1:2000), rabbit anti-BCL2-Associated X Protein (BAX) antibody ( T40051 , Abmart Shanghai Co.,Ltd., China, 1:2000), rabbit anti-BCL2-Associated X Protein (BAX) antibody (ab182858, abcam, Cambridge, UK; 1:2000), and internal control rabbit anti-Tubulin antibody (K006154P, Beijing Solarbio Science & Technology Co.,Ltd., China, 1:2000) overnight at 4°C.

Techniques: Western Blot