Journal: Cell Death & Disease

Article Title: Cell surface nucleolin is a novel ADAMTS5 receptor mediating endothelial cell apoptosis

doi: 10.1038/s41419-022-04618-x

Figure Lengend Snippet: A , B TS5-p45 binds to the HUVEC surface in a dose-dependent manner. Intact HUVECs were treated with various doses of TS5-p45, and surface-bound TS5-p45 was measured through A cellular ELISA by anti-His detection and B flow cytometry by anti-His-FITC detection. IgG, isotype control antibody. Cellular ELISA data shown are mean ± SD from two independent experiments in triplicates. In flow cytometry experiments, anti-IgG-FITC signal was used to set the threshold. The representative set is shown in overlay histogram format. Flow cytometry gating applied for this experiment is shown in Fig. S . C – E NCL directly binds TS5-p45. C TS5-p45 binding partners on HUVEC MF were identified by pulldown for subsequent MS analysis. D Two-way co-IP using TS5-p45 (anti-His) and HUVEC MF showed TS5-p45 and NCL binding. E Two-way co-IP using recombinant TS5-p45 and recombinant NCL demonstrating direct binding between TS5-p45 and NCL. F Confocal microscopy showing co-localization of surface-bound TS5-p45 with endogenous cell surface NCL. Anti-His (TS5-p45, green), anti-NCL (red), and DAPI (Nuc marker, blue) triple staining and the three-planar view of cells were shown. Scale bar represents 5 μm. G siRNA knockdown reduced cell surface expression level of NCL in HUVECs. siNCL-transfected HUVECs were analyzed by anti-NCL WB, with VE-cadherin used as a loading control for MF. WB showed the purity of the MF with anti-Lamin A/C (Nuc marker), anti-HSP60 (Mito marker), and anti-GAPDH (Cyto marker). H , I Cell surface-bound TS5-p45 is reduced upon siNCL knockdown. siRNA-transfected HUVECs were treated with TS5-p45, and cell surface-bound TS5-p45 was measured through H cellular ELISA by anti-His detection and I flow cytometry by anti-His-FITC detection. IgG, isotype control antibody. Cellular ELISA data shown are mean ± SD from two independent experiments in triplicates. In flow cytometry experiments, anti-IgG-FITC signal was used to set the thresholds, respectively. Data from a representative set is shown in separate overlay histograms. Gating applied for this flow cytometry experiment is shown in Fig. S . J , K Determination of binding affinity between NCL and TS5-p45. J SPR sensorgrams from TS5-p45 as analyte over NCL-immobilized chip surface. Red and black curves represent the experimental data and fit to the 1:1 model of the interaction, respectively, with the binding constants indicated. K Binding affinity determined by ELISA. Saturation binding of TS5-p45 to NCL-captured wells was performed. Each treatment concentration/data point is the average of triplicate readings. L NCL is a functional receptor for TS5-p45-induced apoptosis. siRNA-transfected HUVECs with reduced NCL expression were treated with TS5-p45 and apoptosis measured at 24 h post-treatment. Relative apoptosis is normalized to the VEGF-containing condition. Data shown are mean ± SD from three independent experiments in triplicates. Statistical analysis was performed by one-way ANOVA. ns not significant; * p < 0.05; ** p < 0.01; *** p < 0.001 and **** p < 0.0001.

Article Snippet: Recombinant human NCL (FLAG-tagged, full length) was purchased from OriGene (TP319082).

Techniques: Enzyme-linked Immunosorbent Assay, Flow Cytometry, Binding Assay, Co-Immunoprecipitation Assay, Recombinant, Confocal Microscopy, Marker, Staining, Expressing, Transfection, Concentration Assay, Functional Assay

Journal: Cell Death & Disease

Article Title: Cell surface nucleolin is a novel ADAMTS5 receptor mediating endothelial cell apoptosis

doi: 10.1038/s41419-022-04618-x

Figure Lengend Snippet: A Schematic representation of NCL expression constructs with N-terminal FLAG tag encoding NCL-FL 1-710 , NCLΔGAR 1-652 , NCLΔC 1-269 , NCLΔN 270-710 , and NCL-RBD 270-652 . B Expression of NCL and truncates in HEK293T cells by WB via anti-FLAG antibody. GAPDH was used as a loading control. C – G Co-IP analyses of FLAG-tagged NCL and truncates against His-tagged TS5-p45. Binding between TS5-p45 and C NCL-FL 1-710 , D NCLΔGAR 1-652 , E NCLΔC 1-269 , F NCLΔN 270-710 , G NCL-RBD 270-652 were shown by anti-FLAG and anti-His blotting, respectively.

Article Snippet: Recombinant human NCL (FLAG-tagged, full length) was purchased from OriGene (TP319082).

Techniques: Expressing, Construct, FLAG-tag, Co-Immunoprecipitation Assay, Binding Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: Nucleolin regulates the proliferation of vascular smooth muscle cells in atherosclerotic via Aurora B

doi: 10.1111/jcmm.16125

Figure Lengend Snippet: Nucleolin is up‐regulated during atherosclerosis development. (A) Representative immunostaining and quantification for nucleolin on aortic cross sections of ApoE –/– mice fed with chow and high‐fat diets. (Colorimetric images showing nucleolin in brown). Magnification 200×. HFD: high‐fat diet. **, P < .01, vs. control group, n = 3. (B) Immunofluorescence analysis showed co‐localization of nucleolin with SMA + cells in ApoE –/– mice fed with chow and high‐fat diets. Immunostaining for nucleolin(red), SMA (green) and DAPI (blue). (C) Immunofluorescence analysis showed co‐localization of nucleolin with SMA + cells in perivascular carotid collarp placement (PCCP) group and conrol group (n = 3). Immunostaining for nucleolin(red), SMA (green) and DAPI (blue). (D) Immunofluorescence analysis showed Ki67 with SMA + cells in perivascular carotid collarp placement (PCCP) group and conrol group (n = 3). Immunostaining for SMA (red), Ki67 (green) and DAPI (blue)

Article Snippet: Anti‐rabbit secondary antibody (1:10 000 dilution, BA1055, Boster) to detect nucleolin protein and aurora B protein and anti‐mouse secondary antibody (1:2500 dilution, BA1050, Boster) to detect β‐Actin protein.

Techniques: Immunostaining, Control, Immunofluorescence

Journal: Journal of Cellular and Molecular Medicine

Article Title: Nucleolin regulates the proliferation of vascular smooth muscle cells in atherosclerotic via Aurora B

doi: 10.1111/jcmm.16125

Figure Lengend Snippet: POVPC or ox‐LDL up‐regulated nucleolin mRNA and protein expression in HAVSMCs. (A and C) Representative Western blot showing nucleolin levels in VSMCs treated with POVPC or ox‐LDL. *, P < .05, **, P < .01, compared with control, n = 3. (B and D) RT‐qPCR gene expression analysis of VSMCs treated with different stimuli. Unpaired 2‐tailed Student's t test was used to compare means for A‐D. *, P < .05, compared with control, n = 3

Article Snippet: Anti‐rabbit secondary antibody (1:10 000 dilution, BA1055, Boster) to detect nucleolin protein and aurora B protein and anti‐mouse secondary antibody (1:2500 dilution, BA1050, Boster) to detect β‐Actin protein.

Techniques: Expressing, Western Blot, Control, Quantitative RT-PCR, Gene Expression

Journal: Journal of Cellular and Molecular Medicine

Article Title: Nucleolin regulates the proliferation of vascular smooth muscle cells in atherosclerotic via Aurora B

doi: 10.1111/jcmm.16125

Figure Lengend Snippet: The cell proliferation ability changed after interference with the expression of nucleolin. (A) The protein expression of nucleolin fusion protein in vascular smooth muscle cells after transfection with pcDNA3.1‐NCL for 48 hours. The right‐hand side showed the grey ratio analysis of nucleolin/ β‐actin. *, P < .05, compared with vector control (pcDNA3.1), n = 3. (B) The expression of nucleolin protein in vascular smooth muscle cells after transfection with nucleolin specific siRNA. The right‐hand side showed the grey ratio analysis of nucleolin/actin. *, P < .05, compared with negative control (siNC), n = 3. (C) The effect of nucleolin siRNA on cell viability in VSMCs. *, P < .05, vs siNC group, n = 6. (D) The effects of nucleolin overexpression on cell viability in VSMCs. **, P < .01, vs pcDNA3.1 group, n = 6. P ‐values were determined using the two‐tailed Student's t test for comparing two groups and one‐way ANOVA for comparing multiple groups

Article Snippet: Anti‐rabbit secondary antibody (1:10 000 dilution, BA1055, Boster) to detect nucleolin protein and aurora B protein and anti‐mouse secondary antibody (1:2500 dilution, BA1050, Boster) to detect β‐Actin protein.

Techniques: Expressing, Transfection, Plasmid Preparation, Control, Negative Control, Over Expression, Two Tailed Test

Journal: Journal of Cellular and Molecular Medicine

Article Title: Nucleolin regulates the proliferation of vascular smooth muscle cells in atherosclerotic via Aurora B

doi: 10.1111/jcmm.16125

Figure Lengend Snippet: Nucleolin ablation or overexpression change the cell cycle progression of VSMCs. (A) The cell cycle changes in vascular smooth muscle cells after transfected with pcDNA3.1 or pcDNA3.1‐NCL. Percentages of cells in G0/G1, S and G2/M phases after the indicated transfected treatments. Representative flow cytometry graphs are shown, and results are expressed as the mean ± standard deviation from three independent experiments, *, P < .05, vs pcDNA3.1 group (vector control), n = 3. (B) The cell cycle changes in vascular smooth muscle cells after transfected with siNCL or siNC. Percentages of cells in G0/G1, S and G2/M phases after the indicated transfected treatments. *, P < .05, compared with siNC (negative control), n = 3. (C) Representative images of EdU staining in vascular smooth muscle cells after transfected with negative control, pcDNA3.1 or pcDNA3.1‐NCL. EdU‐positive cells increased significantly in pcDNA3.1‐NCL group and the percentage of EdU‐positive cells was calculated, EdU (red) and hoechst (blue), n = 6, *, P < .05, compared with the pcDNA3.1 group. (D) Representative images of EdU staining in vascular smooth muscle cells after transfected with negative control, siNC or siNCL. EdU‐positive cells(red) decreased in siNCL group and the percentage of EdU‐positive cells was calculated, n = 6, *, P < .05, compared with the siNC group. (E) Representative Western blot showing PCNA and Ki67 levels in VSMCs after transfected with pcDNA3.1 or pcDNA3.1‐NCL group. n = 3, *, P < .05, **, P < .01, compared with the pcDNA3.1 group. (F) Representative Western blot showing PCNA and Ki67 levels after transfected with siNC or siNCL group. n = 3, *, P < .05, **, P < .01, compared with the siNC group. P‐values were determined using the two‐tailed Student's t test for comparing two groups and one‐way ANOVA for comparing multiple groups

Article Snippet: Anti‐rabbit secondary antibody (1:10 000 dilution, BA1055, Boster) to detect nucleolin protein and aurora B protein and anti‐mouse secondary antibody (1:2500 dilution, BA1050, Boster) to detect β‐Actin protein.

Techniques: Over Expression, Transfection, Flow Cytometry, Standard Deviation, Plasmid Preparation, Control, Negative Control, Staining, Western Blot, Two Tailed Test

Journal: Journal of Cellular and Molecular Medicine

Article Title: Nucleolin regulates the proliferation of vascular smooth muscle cells in atherosclerotic via Aurora B

doi: 10.1111/jcmm.16125

Figure Lengend Snippet: Aurora B is a direct target of nucleolin in VSMCs. (A) Aurora B as a potential target gene by previous studies and String software ( https://string‐db.org/ ). (B) The protein expression of aurora B fusion protein in vascular smooth muscle cells treated with POVPC or ox‐LDL. n = 3; **, P < .01 vs control group. (C) VSMCs were transfected with pcDNA3.1 or pcDNA3.1‐NCL. The protein level of aurora B was measured by Western blot. n = 3; **, P < .01 vs pcDNA3.1 (vector control). (D) VSMCs were transfected with siNCL or siNC. The protein level of aurora B was measured by Western blot. n = 3; **, P < .01 vs siNC (negative control). (E) Interaction of nucleolin and aurora B tested by immunoprecipitation. Lane 1 represented whole cell lysate. Lane2‐4 represented the proteins precipitated by control IgG, anti‐nucleolin, anti‐aurora B. The upper band indicated nucleolin, and the lower band indicated aurora B. Each experiment was repeated three times. P‐values were determined using the two‐tailed Student's t test for comparing two groups and one‐way ANOVA for comparing multiple groups

Article Snippet: Anti‐rabbit secondary antibody (1:10 000 dilution, BA1055, Boster) to detect nucleolin protein and aurora B protein and anti‐mouse secondary antibody (1:2500 dilution, BA1050, Boster) to detect β‐Actin protein.

Techniques: Software, Expressing, Control, Transfection, Western Blot, Plasmid Preparation, Negative Control, Immunoprecipitation, Two Tailed Test

Journal: Journal of Cellular and Molecular Medicine

Article Title: Nucleolin regulates the proliferation of vascular smooth muscle cells in atherosclerotic via Aurora B

doi: 10.1111/jcmm.16125

Figure Lengend Snippet: Schematic summary of the findings. POVPC or ox‐LDL up‐regulated nucleolin mRNA and protein expression in HAVSMCs, and then the cell cycle changes. Nucleolin may interact with aurora B to regulate the cell cycle and proliferation of vascular smooth muscle cells

Article Snippet: Anti‐rabbit secondary antibody (1:10 000 dilution, BA1055, Boster) to detect nucleolin protein and aurora B protein and anti‐mouse secondary antibody (1:2500 dilution, BA1050, Boster) to detect β‐Actin protein.

Techniques: Expressing

Journal: PLoS ONE

Article Title: Prognostic Significance of the Combined Score of Endothelial Expression of Nucleolin and CD31 in Surgically Resected Non-Small Cell Lung Cancer

doi: 10.1371/journal.pone.0054674

Figure Lengend Snippet: The tumor blood vessels and nucleolin were stained with anti-CD31 (green) and anti-nucleolin (red), respectively. Nuclei were stained with DAPI (blue). Representative images from the three groups were shown here. (A) CD31 hi NCL hi , (B) CD31 hi NCL lo , (C) CD31 lo NCL lo . Scale bar, 50 µm.

Article Snippet: According to methods previously described , , the deparaffinized sections were incubated at 4°C overnight with mouse anti-human CD31 mAb (Santa Cruz Biotechnologies, Santa Cruz, CA, USA) and rabbit anti-human nucleolin pAb (Protgen, Beijing, China).

Techniques: Staining

Journal: PLoS ONE

Article Title: Prognostic Significance of the Combined Score of Endothelial Expression of Nucleolin and CD31 in Surgically Resected Non-Small Cell Lung Cancer

doi: 10.1371/journal.pone.0054674

Figure Lengend Snippet: Univariate analyses of DFS and OS according to the expression of CD31 and nucleolin in all patients and subgroups with clinicopathologic variables (Log-rank test).

Article Snippet: According to methods previously described , , the deparaffinized sections were incubated at 4°C overnight with mouse anti-human CD31 mAb (Santa Cruz Biotechnologies, Santa Cruz, CA, USA) and rabbit anti-human nucleolin pAb (Protgen, Beijing, China).

Techniques: Expressing

Journal: PLoS ONE

Article Title: Prognostic Significance of the Combined Score of Endothelial Expression of Nucleolin and CD31 in Surgically Resected Non-Small Cell Lung Cancer

doi: 10.1371/journal.pone.0054674

Figure Lengend Snippet: Multivariate Cox’s proportional hazards model analyses of survival and bone metastasis (BMT).

Article Snippet: According to methods previously described , , the deparaffinized sections were incubated at 4°C overnight with mouse anti-human CD31 mAb (Santa Cruz Biotechnologies, Santa Cruz, CA, USA) and rabbit anti-human nucleolin pAb (Protgen, Beijing, China).

Techniques: