anti icam1 antibodies  (R&D Systems)

Journal: Frontiers in Immunology

Article Title: CT-based radiogenomic prediction of ICAM1 and RAET1E as biomarkers of NK cytotoxicity in clear cell renal cell carcinoma

doi: 10.3389/fimmu.2026.1773251

Figure Lengend Snippet: Expression patterns and prognostic associations of ICAM1 and RAET1E in ccRCC. (A) Representative immunohistochemical staining showing high ICAM1 expression in ccRCC tissues. (B) Violin plot showing the positive association between ICAM1 expression and tumor stage. (C) Kaplan–Meier survival analysis showing that high ICAM1 expression was associated with poorer overall survival. (D) Representative immunohistochemical staining showing low RAET1E expression in ccRCC tissues. (E) Violin plot showing the negative association between RAET1E expression and tumor stage. (F) Kaplan–Meier survival analysis showing that high RAET1E expression was associated with improved overall survival.

Article Snippet: Tissue sections were incubated overnight at 4 °C with anti-N2DL4 monoclonal antibody (RAET1E-encoded protein; MA5-24328, Invitrogen; 1:80) or anti-ICAM1 monoclonal antibody (GB14040, Servicebio; 1:200).

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Frontiers in Immunology

Article Title: CT-based radiogenomic prediction of ICAM1 and RAET1E as biomarkers of NK cytotoxicity in clear cell renal cell carcinoma

doi: 10.3389/fimmu.2026.1773251

Figure Lengend Snippet: Stage-dependent expression and clinical associations of ICAM1 in ccRCC. (A) Representative immunohistochemical staining of ICAM1 across WHO/ISUP stages I–IV, showing progressively increased expression in advanced tumors. (B) Violin plot showing the positive association between ICAM1 expression and tumor stage. (C) Kaplan–Meier survival analysis indicating poorer overall survival in patients with high ICAM1 expression. (D) Distribution of ICAM1 staining categories stratified by expression (<50 and ≥50).

Article Snippet: Tissue sections were incubated overnight at 4 °C with anti-N2DL4 monoclonal antibody (RAET1E-encoded protein; MA5-24328, Invitrogen; 1:80) or anti-ICAM1 monoclonal antibody (GB14040, Servicebio; 1:200).

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Frontiers in Immunology

Article Title: CT-based radiogenomic prediction of ICAM1 and RAET1E as biomarkers of NK cytotoxicity in clear cell renal cell carcinoma

doi: 10.3389/fimmu.2026.1773251

Figure Lengend Snippet: Immune deconvolution analysis of ICAM1 and RAET1E in TCGA-KIRC. (A) Correlation between ICAM1 expression and inferred NK-cell abundance estimated by MCP-counter. (B) Correlation between RAET1E expression and inferred NK-cell abundance (C) Comparison of NK-cell abundance between ICAM1-high and ICAM1-low tumors. (D) Comparison of NK-cell abundance between RAET1E-high and RAET1E-low tumors. Both ICAM1 and RAET1E expression showed significant positive associations with inferred NK-cell abundance.

Article Snippet: Tissue sections were incubated overnight at 4 °C with anti-N2DL4 monoclonal antibody (RAET1E-encoded protein; MA5-24328, Invitrogen; 1:80) or anti-ICAM1 monoclonal antibody (GB14040, Servicebio; 1:200).

Techniques: Expressing, Comparison

Journal: Frontiers in Immunology

Article Title: CT-based radiogenomic prediction of ICAM1 and RAET1E as biomarkers of NK cytotoxicity in clear cell renal cell carcinoma

doi: 10.3389/fimmu.2026.1773251

Figure Lengend Snippet: Combined ICAM1/RAET1E stratification reveals immune-context and clinical heterogeneity in TCGA-KIRC. (A) Four-group stratification based on median ICAM1 and RAET1E expression. (B) Comparison of inferred NK-cell abundance across the four groups. (C) Comparison of KEGG NK cytotoxicity pathway scores across the four groups. (D) Distribution of pathological stage across the four groups. (E) Kaplan–Meier survival analysis among the four subgroups. (F) Forest plot comparing the ICAM1-high/RAET1E-low subgroup with all remaining tumors combined. The ICAM1-high/RAET1E-low subgroup showed an unfavorable survival association relative to the remaining tumors.

Article Snippet: Tissue sections were incubated overnight at 4 °C with anti-N2DL4 monoclonal antibody (RAET1E-encoded protein; MA5-24328, Invitrogen; 1:80) or anti-ICAM1 monoclonal antibody (GB14040, Servicebio; 1:200).

Techniques: Expressing, Comparison

Journal: eLife

Article Title: Clathrin-independent endocytosis and retrograde transport in cancer cells tune immune synapse organization and CD8 T cell response

doi: 10.7554/eLife.105821

Figure Lengend Snippet: HeLa cells stably expressing EndoA3-GFP (green) and transiently expressing ICAM1-mScarlet (red) were imaged for 2 min using live-cell TIRF microscopy, with 1-s intervals between frames. Representative of two independent experiments. Scale bar: 10 µm.

Article Snippet: First, anti-ALCAM (Bio-Rad, MCA1926) and anti-ICAM1 (Bio-Rad, MCA1615) antibodies were labeled with benzylguanine (BG) by incubating them overnight at 4°C with a threefold molar excess of BG-GLA-NHS reagent (New England Biolabs, S9151S; prepared in anhydrous DMSO).

Techniques:

Journal: eLife

Article Title: Clathrin-independent endocytosis and retrograde transport in cancer cells tune immune synapse organization and CD8 T cell response

doi: 10.7554/eLife.105821

Figure Lengend Snippet: LB33-MEL cells stably expressing EndoA3-GFP (green) and transiently expressing ICAM1-mScarlet (red) were imaged for 2 min using live-cell TIRF microscopy, with 1-s intervals between frames. Representative of two independent experiments. Scale bars: 10 μm (full-size image) and 2 μm (enlarged cropped area).

Article Snippet: First, anti-ALCAM (Bio-Rad, MCA1926) and anti-ICAM1 (Bio-Rad, MCA1615) antibodies were labeled with benzylguanine (BG) by incubating them overnight at 4°C with a threefold molar excess of BG-GLA-NHS reagent (New England Biolabs, S9151S; prepared in anhydrous DMSO).

Techniques:

Journal: eLife

Article Title: Clathrin-independent endocytosis and retrograde transport in cancer cells tune immune synapse organization and CD8 T cell response

doi: 10.7554/eLife.105821

Figure Lengend Snippet: HeLa cells stably expressing EndoA3-GFP (green) and transiently expressing ICAM1-mScarlet (red) were imaged for 2 min using live-cell TIRF microscopy, with 1-s intervals between frames. The punctate ICAM1 structure is indicated by a white arrowhead. Representative of two independent experiments. Scale bar: 10 µm.

Article Snippet: First, anti-ALCAM (Bio-Rad, MCA1926) and anti-ICAM1 (Bio-Rad, MCA1615) antibodies were labeled with benzylguanine (BG) by incubating them overnight at 4°C with a threefold molar excess of BG-GLA-NHS reagent (New England Biolabs, S9151S; prepared in anhydrous DMSO).

Techniques:

Journal: eLife

Article Title: Clathrin-independent endocytosis and retrograde transport in cancer cells tune immune synapse organization and CD8 T cell response

doi: 10.7554/eLife.105821

Figure Lengend Snippet: LB33-MEL cells stably expressing EndoA3-GFP (green) and transiently expressing ICAM1-mScarlet (red) were imaged for 2 min using live-cell TIRF microscopy, with 1-s intervals between frames. The punctate ICAM1 structure is indicated by a white arrowhead. Representative of two independent experiments. Scale bars: 10 μm (full-size image) and 2 μm (enlarged cropped area).

Article Snippet: First, anti-ALCAM (Bio-Rad, MCA1926) and anti-ICAM1 (Bio-Rad, MCA1615) antibodies were labeled with benzylguanine (BG) by incubating them overnight at 4°C with a threefold molar excess of BG-GLA-NHS reagent (New England Biolabs, S9151S; prepared in anhydrous DMSO).

Techniques:

Journal: eLife

Article Title: Clathrin-independent endocytosis and retrograde transport in cancer cells tune immune synapse organization and CD8 T cell response

doi: 10.7554/eLife.105821

Figure Lengend Snippet: HeLa cells stably expressing EndoA3-GFP (green) and transiently expressing ICAM1-mScarlet (red) were imaged for 2 min using live-cell TIRF microscopy, with 1-s intervals between frames. Representative of two independent experiments. Scale bars: 10 μm (full-size image) and 2 μm (enlarged cropped area).

Article Snippet: First, anti-ALCAM (Bio-Rad, MCA1926) and anti-ICAM1 (Bio-Rad, MCA1615) antibodies were labeled with benzylguanine (BG) by incubating them overnight at 4°C with a threefold molar excess of BG-GLA-NHS reagent (New England Biolabs, S9151S; prepared in anhydrous DMSO).

Techniques:

Journal: eLife

Article Title: Clathrin-independent endocytosis and retrograde transport in cancer cells tune immune synapse organization and CD8 T cell response

doi: 10.7554/eLife.105821

Figure Lengend Snippet: LB33-MEL cells stably expressing EndoA3-GFP (green) and transiently expressing ICAM1-mScarlet (red) were imaged for 2 min using live-cell TIRF microscopy, with 1-s intervals between frames. Representative of two independent experiments. Scale bar: 10 µm.

Article Snippet: First, anti-ALCAM (Bio-Rad, MCA1926) and anti-ICAM1 (Bio-Rad, MCA1615) antibodies were labeled with benzylguanine (BG) by incubating them overnight at 4°C with a threefold molar excess of BG-GLA-NHS reagent (New England Biolabs, S9151S; prepared in anhydrous DMSO).

Techniques:

Journal: eLife

Article Title: Clathrin-independent endocytosis and retrograde transport in cancer cells tune immune synapse organization and CD8 T cell response

doi: 10.7554/eLife.105821

Figure Lengend Snippet: LB33-MEL cells stably expressing EndoA3-GFP (green) and transiently expressing ICAM1-mScarlet (red) were imaged for 2 min using live-cell TIRF microscopy, with 1-s intervals between frames. The punctate ICAM1 structure is indicated by a white arrowhead. Representative of two independent experiments. Scale bar: 10 µm.

Article Snippet: First, anti-ALCAM (Bio-Rad, MCA1926) and anti-ICAM1 (Bio-Rad, MCA1615) antibodies were labeled with benzylguanine (BG) by incubating them overnight at 4°C with a threefold molar excess of BG-GLA-NHS reagent (New England Biolabs, S9151S; prepared in anhydrous DMSO).

Techniques:

Journal: eLife

Article Title: Clathrin-independent endocytosis and retrograde transport in cancer cells tune immune synapse organization and CD8 T cell response

doi: 10.7554/eLife.105821

Figure Lengend Snippet: A HeLa cell transiently expressing ICAM1-EGFP (green) forming an immune synapse-like conjugate with a CD8 T cell (red) was imaged by live-cell spinning-disk confocal microscopy, for 3 min, with 1-s intervals between frames. ICAM1-positive tubulo-vesicular carriers are observed fusing at the ICAM1-enriched contact zone, which grows and expands over time. Notably, anterograde transport of ICAM1-positive carriers toward the contact zone appears stronger than retrograde transport, and carrier density is higher in the region of the HeLa cell engaged in the immune synapse compared with regions not involved in synapse formation. Associated tracking and quantification data are shown in Figure 4C–G . Representative of two independent experiments. Scale bar: 20 µm.

Article Snippet: First, anti-ALCAM (Bio-Rad, MCA1926) and anti-ICAM1 (Bio-Rad, MCA1615) antibodies were labeled with benzylguanine (BG) by incubating them overnight at 4°C with a threefold molar excess of BG-GLA-NHS reagent (New England Biolabs, S9151S; prepared in anhydrous DMSO).

Techniques:

Journal: eLife

Article Title: Clathrin-independent endocytosis and retrograde transport in cancer cells tune immune synapse organization and CD8 T cell response

doi: 10.7554/eLife.105821

Figure Lengend Snippet: A HeLa cell transiently expressing ICAM1-EGFP (green) forming an immune synapse-like conjugate with a CD8 T cell (red) was imaged by live-cell spinning-disk confocal microscopy, for 3 min, with 1-s intervals between frames. ICAM1-positive tubulo-vesicular carriers are observed fusing at the ICAM1-enriched contact zone (white arrows), which grows and expands over time. Representative of two independent experiments. Scale bar: 5 µm.

Article Snippet: First, anti-ALCAM (Bio-Rad, MCA1926) and anti-ICAM1 (Bio-Rad, MCA1615) antibodies were labeled with benzylguanine (BG) by incubating them overnight at 4°C with a threefold molar excess of BG-GLA-NHS reagent (New England Biolabs, S9151S; prepared in anhydrous DMSO).

Techniques:

Journal: eLife

Article Title: Clathrin-independent endocytosis and retrograde transport in cancer cells tune immune synapse organization and CD8 T cell response

doi: 10.7554/eLife.105821

Figure Lengend Snippet: ( A ) Illustration of the SNAP-tag-based BG-labeled antibody uptake assay to study membrane protein endocytosis and retrograde transport. ( B ) Confocal images of GalT-GFP-SNAP (green) and TMR-Star (red) in HeLa cells stably expressing the Golgi-resident GFP-fused SNAP-tag construct (HeLa GalT-GFP-SNAP). Actin (phalloidin, white) and nuclei (DAPI, blue) were also stained. Fluorescence intensity profile was made along the dashed line region in enlarged cropped area and shows the colocalization of both signals. Scale bar: 20 μm. ( C–F ) Retrograde transport of ALCAM and ICAM1. Continuous BG-labeled anti-ALCAM ( C, E ) and anti-ICAM1 ( D, F ) antibody uptake for 4 h at 37°C in HeLa GalT-GFP-SNAP cells. ( C, D ) Western blot analysis of HeLa GalT-GFP-SNAP cells transfected for 72 h with siRNAs: negative control (siCtrl) or against retromer subunits (siVPS35 and siVPS26A). Immunodetection made with anti-SNAP, anti-VPS35, anti-VPS26A, and anti-α-Tubulin (loading control) antibodies. Quantification of the covalent IgG-SNAP-GFP-GalT complex is shown as fractions of siCtrl condition (histogram). Quantification of VPS35 and VPS26A depletion is shown in . ( E, F ) Western blot analysis of HeLa GalT-GFP-SNAP cells transfected for 72 h with siRNAs: negative control (siCtrl) or against EndoA3 (siEndoA3). Immunodetection made with anti-SNAP, anti-EndoA3, and anti-clathrin heavy chain (CHC, loading control) antibodies. Quantification of the covalent IgG-SNAP-GFP-GalT complex (IB:anti-SNAP) is shown as fractions of siCtrl condition (histogram). Quantification of EndoA3 depletion is shown in . Data information: In ( B ), images are from a single experiment. Quantification data ( C–F ) are pooled from three independent experiments. Data are presented as mean ± SEM. *p<0.05, **p<0.01. One-sample t test and Wilcoxon test. Figure 1—source data 1. Original files for western blot analyses displayed in . Figure 1—source data 2. PDF files containing original western blots for .

Article Snippet: First, anti-ALCAM (Bio-Rad, MCA1926) and anti-ICAM1 (Bio-Rad, MCA1615) antibodies were labeled with benzylguanine (BG) by incubating them overnight at 4°C with a threefold molar excess of BG-GLA-NHS reagent (New England Biolabs, S9151S; prepared in anhydrous DMSO).

Techniques: Labeling, Membrane, Stable Transfection, Expressing, Construct, Staining, Fluorescence, Western Blot, Transfection, Negative Control, Immunodetection, Control

Journal: eLife

Article Title: Clathrin-independent endocytosis and retrograde transport in cancer cells tune immune synapse organization and CD8 T cell response

doi: 10.7554/eLife.105821

Figure Lengend Snippet: ( A ) Confocal images of GalT-GFP-SNAP (green) and TMR-Star (red) in LB33-MEL cells stably expressing the Golgi-resident GFP-fused SNAP-tag construct (LB33-MEL GalT-GFP-SNAP). Actin (phalloidin, white) and nuclei (DAPI, blue) were also stained. The fluorescence intensity profile was made along the dashed line region in the enlarged cropped area and shows the colocalization of both signals. Scale bar: 20 μm. ( B ) Confocal images of GalT-GFP-SNAP (green) and TGN46 (red) in LB33-MEL GalT-GFP-SNAP cells. Nuclei (DAPI, blue) were also stained. The fluorescence intensity profile was made along the dashed line region in enlarged cropped area and shows the colocalization of both signals, indicating that GalT-GFP-SNAP is correctly localized at the TGN . Scale bar: 10 μm. ( C ) Quantifications of the immunoblots shown in (IB: anti-VPS35 and anti-VPS26A) confirm depletion efficiency of VPS35 and VPS26A in HeLa GalT-GFP-SNAP cells. ( D ) Retrograde transport of ICAM-1. Continuous BG-labeled anti-ICAM1 antibody uptake for 4 h at 37°C in LB33-MEL GalT-GFP-SNAP cells. Western blot analysis of LB33-MEL GalT-GFP-SNAP cells transfected with siRNAs: negative control (siCtrl) or against retromer subunits (siVPS35 and siVPS26A). Immunodetection with anti-SNAP, anti-VPS35, anti-VPS26A, and anti-α-Tubulin (loading control) antibodies. Quantification of the covalent IgG-SNAP complex is shown as fractions of siCtrl condition (histogram). Quantification of VPS35 and VPS26A depletion is shown in ( E ). ( E ) Quantifications of the immunoblots shown in ( D ) confirm depletion efficiency of VPS35 and VPS26A in LB33-MEL GalT-GFP-SNAP cells. ( F ) Retrograde transport of ALCAM. Continuous BG-labeled anti-ALCAM antibody uptake for 4 h at 37°C in HeLa GalT-GFP-SNAP cells. Western blot analysis of HeLa GalT-GFP-SNAP cells transfected for 72 h with siRNAs: negative control (siCtrl) or against Rab6 (siRab6). Immunodetection made with anti-SNAP, anti-Rab6, and anti-clathrin heavy chain (CHC, loading control) antibodies. Quantification of the covalent IgG-SNAP-GFP-GalT complex is shown as fractions of siCtrl condition (histogram). Quantification of Rab6 depletion is shown in ( G ). ( G ) Quantifications of the immunoblots shown in ( F ) confirm depletion efficiency of Rab6 in HeLa GalT-GFP-SNAP cells. ( H ) Quantifications of the immunoblots shown in (IB: anti-EndoA3) confirm depletion efficiency of EndoA3 in HeLa GalT-GFP-SNAP cells. ( I ) Retrograde transport of ALCAM. Continuous BG-labeled anti-ALCAM antibody uptake for 4 h at 37°C in LB33-MEL GalT-GFP-SNAP cells. Western blot analysis of LB33-MEL GalT-GFP-SNAP cells transfected (or not) with plasmids encoding free GFP (GFP+) or EndoA3-GFP (EndoA3+). Immunodetection with anti-SNAP, anti-EndoA3, and anti-α-Tubulin (loading control) antibodies. Quantification of the covalent IgG-SNAP-GFP-GalT complex is shown as fractions of the GFP +condition (histogram). Data information: In ( A, B ), images are from a single experiment. In ( C ), data are pooled from six independent experiments. In ( D–H ), data are pooled from three independent experiments. In ( I ), data are pooled from two independent experiments. Data are presented as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. One-sample t test and Wilcoxon test. Figure 1—figure supplement 1—source data 1. Original files for western blot analyses displayed in . Figure 1—figure supplement 1—source data 2. PDF files containing original western blots for .

Article Snippet: First, anti-ALCAM (Bio-Rad, MCA1926) and anti-ICAM1 (Bio-Rad, MCA1615) antibodies were labeled with benzylguanine (BG) by incubating them overnight at 4°C with a threefold molar excess of BG-GLA-NHS reagent (New England Biolabs, S9151S; prepared in anhydrous DMSO).

Techniques: Stable Transfection, Expressing, Construct, Staining, Fluorescence, Western Blot, Labeling, Transfection, Negative Control, Immunodetection, Control

Journal: Journal of Nanobiotechnology

Article Title: COPD airway epithelial cells–derived extracellular vesicles contribute to endothelial dysfunction and atherosclerosis via the miR-141-3p/PDCD4 axis

doi: 10.1186/s12951-026-04091-0

Figure Lengend Snippet: COPD Airway Epithelium Derived-EVs Promote Endothelial Inflammation and Apoptosis. ( A ) Non-COPD- and COPD-derived EVs were labeled with PKH26 and incubated with HUVECs. After 24 h, HUVECs were washed, fixed, and counterstained with DAPI. Representative confocal images show the internalization of primary human AECs-derived EVs by HUVECs. Scale bar: 20 μm. (B–G) Representative Western blot images ( B ) and quantification ( C–G ) of VCAM1, ICAM1, BAX, BCL2, Cleaved Caspase-3/Caspase-3, and GAPDH in TNFα-pretreated HUVECs incubated with PBS, Non-COPD EVs, or COPD EVs for 24 h ( n = 6 biological replicates). ( H ) and ( I ) Relative mRNA levels IL-1β and IL-6 in HUVECs describe as above ( n = 6 biological replicates). ( J , K ) TNFα-pretreated HUVECs were incubated with PBS, Non-COPD EVs, or COPD EVs for 24 h and subsequently cocultured with THP-1 cells for 6 h. THP-1 cells were stained with Calcein-AM (green) to visualize adhesion ( J ), and monocyte adhesion was quantified relative to control ( K ) ( n = 6 biological replicates). Scale bar: 100 μm. ( L, M ) Flow cytometric analysis of apoptosis by Annexin V-FITC/PI staining in HUVECs describe as above. Representative dot plots ( L ) and quantification of apoptotic cells ( M ) are shown ( n = 6 biological replicates). Data are presented as mean±SD. Statistical analyses were carried out via one-way ANOVA followed by Bonferroni’s post hoc test. * P < 0.05, ** P < 0.01

Article Snippet: Membranes were blocked with 5% bovine serum albumin for 1 h at room temperature and incubated overnight at 4 °C with the following primary antibodies: CD9 (20597-1-AP, Proteintech, China), ALIX (12422-1-AP, Proteintech, China), CD63 (25682-1-AP, Proteintech, China), Calnexin (2679, CST, USA), Albumin (82783-6-RR, Proteintech, China), ApoA1 (14427-1-AP, Proteintech, China), VCAM1 (ab134047, Abcam, USA), ICAM1 (sc-8439, Santa Cruz, USA), BAX (50599-2-Ig, Proteintech, China), BCL2 (T40056S, Abmart, China), Caspase 3 (T40044S, Abmart, China), Cleaved Caspase 3 (TA7022S, Abmart, China), and GAPDH (2118 S, CST, USA).

Techniques: Derivative Assay, Labeling, Incubation, Western Blot, Staining, Control

Journal: Journal of Nanobiotechnology

Article Title: COPD airway epithelial cells–derived extracellular vesicles contribute to endothelial dysfunction and atherosclerosis via the miR-141-3p/PDCD4 axis

doi: 10.1186/s12951-026-04091-0

Figure Lengend Snippet: miR-141-3p is downregulated in COPD AEC-derived EVs and plays a regulatory role in inflammation and apoptosis of HUVECs. ( A ) Heatmap showing qPCR expression profiles of selected miRNAs in EVs derived from non-COPD and COPD individuals. miRNAs enclosed in red boxes denote significant differences between groups ( P < 0.05). ( B ) The expression levels of miR-141-3p in plasma-derived EVs from non-COPD and COPD participants. ( C-H ) Representative Western blot images ( C ) and quantification ( D–H ) of VCAM1, ICAM1, BAX, BCL2, Cleaved Caspase-3/Caspase-3, and GAPDH expression in HUVECs transfected with ctrl mimics or miR-141-3p mimics with or without TNF-α pre-treatment for 24 h ( n = 3 biological replicates). ( I ) and ( J ) Relative mRNA levels IL-1β and IL-6 in HUVECs describe as above ( n = 3 biological replicates). ( K , L ) Monocyte-endothelial adhesion assay were performed in HUVECs describe as above. Scale bar, 100 μm ( K ). Monocyte adhesion was quantified relative to control ( L ) ( n = 3 biological replicates). ( M , N ) Flow cytometric analysis of apoptosis by Annexin V-FITC/PI staining in HUVECs describe as above. Representative dot plots ( M ) and quantification of apoptotic cells ( N ) are shown ( n = 3 biological replicates). ( O-T ) Representative Western blot images ( O ) and quantification ( P–T ) of VCAM1, ICAM1, BAX, BCL2, Cleaved Caspase-3/Caspase-3, and GAPDH expression in HUVECs transfected with ctrl inhibitors or miR-141-3p inhibitors with or without TNF-α pre-treatment for 24 h ( n = 3 biological replicates). ( U ) and ( V ) Relative mRNA levels IL-1β and IL-6 in HUVECs describe as above ( n = 3 biological replicates). ( W , Y ) Monocyte-endothelial adhesion assay were performed in HUVECs describe as above. Scale bar, 100 μm ( W ). Monocyte adhesion was quantified relative to control (X ) ( n = 3 biological replicates). ( X , Z ) Flow cytometric analysis of apoptosis by Annexin V-FITC/PI staining in HUVECs describe as above. Representative dot plots ( X ) and quantification of apoptotic cells ( Z ) are shown ( n = 3 biological replicates). Data are presented as mean±SD. Statistical analyses were carried out via one-way ANOVA followed by Bonferroni’s post hoc test. * P < 0.05, ** P < 0.01

Article Snippet: Membranes were blocked with 5% bovine serum albumin for 1 h at room temperature and incubated overnight at 4 °C with the following primary antibodies: CD9 (20597-1-AP, Proteintech, China), ALIX (12422-1-AP, Proteintech, China), CD63 (25682-1-AP, Proteintech, China), Calnexin (2679, CST, USA), Albumin (82783-6-RR, Proteintech, China), ApoA1 (14427-1-AP, Proteintech, China), VCAM1 (ab134047, Abcam, USA), ICAM1 (sc-8439, Santa Cruz, USA), BAX (50599-2-Ig, Proteintech, China), BCL2 (T40056S, Abmart, China), Caspase 3 (T40044S, Abmart, China), Cleaved Caspase 3 (TA7022S, Abmart, China), and GAPDH (2118 S, CST, USA).

Techniques: Derivative Assay, Expressing, Clinical Proteomics, Western Blot, Transfection, Cell Adhesion Assay, Control, Staining

Journal: Journal of Nanobiotechnology

Article Title: COPD airway epithelial cells–derived extracellular vesicles contribute to endothelial dysfunction and atherosclerosis via the miR-141-3p/PDCD4 axis

doi: 10.1186/s12951-026-04091-0

Figure Lengend Snippet: EVs-derived miR-141-3p from primary AECs regulates inflammation and apoptosis in HUVECs. ( A ) Relative expression of miR-141-3p in EVs isolated from primary AECs transfected with miR-141-3p mimics or control, measured by qPCR ( n = 3 biological replicates). ( B ) Relative expression of miR-141-3p in HUVECs co-cultured with EVs derived from primary AECs transfected with miR-141-3p mimics (miR-141-3p mimics–EVs) or control, measured by qPCR ( n = 3 biological replicates). ( C , D ) Representative Western blot images ( C ) and quantification ( D ) of VCAM1, ICAM1, BAX, BCL2, Cleaved Caspase-3/Caspase-3, and GAPDH expression in HUVECs co-cultured with ctrl mimics-EVs or miR-141-3p mimics-EVs with TNF-α pre-treatment for 24 h ( n = 3 biological replicates). ( E ) and ( F ) Relative mRNA levels IL-6 and IL-1β in HUVECs describe as above ( n = 3 biological replicates). ( G , H ) Monocyte-endothelial adhesion assay were performed in HUVECs describe as above. Scale bar, 100 μm ( G ). Monocyte adhesion was quantified relative to control ( H ) ( n = 3 biological replicates). ( I , J ) Flow cytometric analysis of apoptosis by Annexin V-FITC/PI staining in HUVECs describe as above. Representative dot plots ( I ) and quantification of apoptotic cells ( J ) are shown ( n = 3 biological replicates). ( K ) Relative expression of miR-141-3p in EVs isolated from primary AECs transfected with miR-141-3p inhibitors or control, measured by qPCR ( n = 3 biological replicates). ( L ) Relative expression of miR-141-3p in HUVECs co-cultured with EVs derived from primary AECs transfected with miR-141-3p inhibitors (miR-141-3p inhibitors–EVs) or control, measured by qPCR ( n = 3 biological replicates). ( M, N ) Representative Western blot images ( M ) and quantification ( N ) of VCAM1, ICAM1, BAX, BCL2, Cleaved Caspase-3/Caspase-3, and GAPDH expression in HUVECs co-cultured with ctrl inhibitors-EVs or miR-141-3p inhibitors-EVs with TNF-α pre-treatment for 24 h ( n = 3 biological replicates). ( O ) and ( P ) Relative mRNA levels IL-6 and IL-1β in HUVECs describe as above ( n = 3 biological replicates). ( Q , R ) Monocyte-endothelial adhesion assay were performed in HUVECs describe as above. Scale bar, 100 μm ( Q ). Monocyte adhesion was quantified relative to control ( R ) ( n = 3 biological replicates). ( S , T ) Flow cytometric analysis of apoptosis by Annexin V-FITC/PI staining in HUVECs describe as above. Representative dot plots ( S ) and quantification of apoptotic cells ( T ) are shown ( n = 3 biological replicates). Data are presented as mean±SD. P -values were calculated using Student’s t-test. * P < 0.05, ** P < 0.01

Article Snippet: Membranes were blocked with 5% bovine serum albumin for 1 h at room temperature and incubated overnight at 4 °C with the following primary antibodies: CD9 (20597-1-AP, Proteintech, China), ALIX (12422-1-AP, Proteintech, China), CD63 (25682-1-AP, Proteintech, China), Calnexin (2679, CST, USA), Albumin (82783-6-RR, Proteintech, China), ApoA1 (14427-1-AP, Proteintech, China), VCAM1 (ab134047, Abcam, USA), ICAM1 (sc-8439, Santa Cruz, USA), BAX (50599-2-Ig, Proteintech, China), BCL2 (T40056S, Abmart, China), Caspase 3 (T40044S, Abmart, China), Cleaved Caspase 3 (TA7022S, Abmart, China), and GAPDH (2118 S, CST, USA).

Techniques: Derivative Assay, Expressing, Isolation, Transfection, Control, Cell Culture, Western Blot, Cell Adhesion Assay, Staining

Journal: Journal of Nanobiotechnology

Article Title: COPD airway epithelial cells–derived extracellular vesicles contribute to endothelial dysfunction and atherosclerosis via the miR-141-3p/PDCD4 axis

doi: 10.1186/s12951-026-04091-0

Figure Lengend Snippet: PDCD4 is involved in the regulation of endothelial inflammation and apoptosis by miR-141-3p. ( A ) Potential target genes of miR-141-3p were identified using TargetScan, miRDB, miRWalk, and PicTar, in combination with apoptosis-related gene sets from the GSEA database. ( B ) Predicted miR-141-3p binding sites in the human and mouse PDCD4 3’UTR, with sequences of WT and mutant reporters shown. ( C , D ) Responsiveness of WT and mutant PDCD4/Pdcd4 3’UTR reporters to miR-141-3p in stably transfected cell lines ( n = 3 biological replicates). ( E ) Relative mRNA levels of PDCD4 in 293 T transfected with miRNA-NC mimics, miRNA-141-3p mimics, miRNA-NC inhibitors or miRNA-141-3p inhibitors ( n = 3 biological replicates). ( H-P ) Representative Western blot images ( H ) and quantification ( I-P ) of VCAM1, ICAM1, BAX, BCL2, Cleaved Caspase-3/Caspase-3, p-P65/P65, and GAPDH expression in HUVECs transfected with siNC/PDCD4 or NC/PDCD4 vector with TNF-α pre-treatment for 24 h ( n = 3 biological replicates). ( N ) and ( O ) Relative mRNA levels IL-6 and IL-1β in HUVECs describe as above ( n = 3 biological replicates). ( Q , R ) Monocyte-endothelial adhesion assay were performed in HUVECs describe as above. Scale bar, 100 μm ( Q ). Monocyte adhesion was quantified relative to control ( R ) ( n = 3 biological replicates). ( S , T ) Flow cytometric analysis of apoptosis by Annexin V-FITC/PI staining in HUVECs describe as above. Representative dot plots ( S ) and quantification of apoptotic cells ( T ) are shown ( n = 3 biological replicates). Statistical analyses were performed using unpaired two-tailed Student’s t-test for comparisons between two groups (siNC vs. siPDCD4 or NC vector vs. PDCD4 vector), as indicated. Data are presented as mean±SD.* P < 0.05, ** P < 0.01

Article Snippet: Membranes were blocked with 5% bovine serum albumin for 1 h at room temperature and incubated overnight at 4 °C with the following primary antibodies: CD9 (20597-1-AP, Proteintech, China), ALIX (12422-1-AP, Proteintech, China), CD63 (25682-1-AP, Proteintech, China), Calnexin (2679, CST, USA), Albumin (82783-6-RR, Proteintech, China), ApoA1 (14427-1-AP, Proteintech, China), VCAM1 (ab134047, Abcam, USA), ICAM1 (sc-8439, Santa Cruz, USA), BAX (50599-2-Ig, Proteintech, China), BCL2 (T40056S, Abmart, China), Caspase 3 (T40044S, Abmart, China), Cleaved Caspase 3 (TA7022S, Abmart, China), and GAPDH (2118 S, CST, USA).

Techniques: Binding Assay, Mutagenesis, Stable Transfection, Transfection, Western Blot, Expressing, Plasmid Preparation, Cell Adhesion Assay, Control, Staining, Two Tailed Test

Journal: Journal of Nanobiotechnology

Article Title: COPD airway epithelial cells–derived extracellular vesicles contribute to endothelial dysfunction and atherosclerosis via the miR-141-3p/PDCD4 axis

doi: 10.1186/s12951-026-04091-0

Figure Lengend Snippet: COPD AECs-Derived EVs Accelerates Atherosclerosis and Triggers Vascular Inflammation and Apoptosis in ApoE -/- Mice. ( A ) Representative fluorescence images of EV distribution in mice 24 h after injection of PBS, DiR, DiR-labeled non-COPD or COPD EVs. ( B ) Ex vivo imaging of major organs, including the heart, liver, spleen, lung, kidney, and aorta. ( C ) Experimental scheme: ApoE -/- Mice fed a high-fat diet and were injected with PBS, Non-COPD or COPD-EVs. ( D ) Representative en face Oil Red O–stained aortas and ( E ) corresponding quantification from ApoE -/- mice fed a high-fat diet and treated with PBS, non-COPD EVs, or COPD EVs ( n = 6 biological replicates). ( F ) Representative images of the thoracic aorta and carotid artery from the three groups after in vivo perfusion with 4% paraformaldehyde. White areas indicate arterial plaques. ( G-T ) Representative images and quantifications of Oil Red O staining ( G , H ), H&E staining (lesion area) ( I , J ), immunofluorescent staining of VCAM1 ( K , L ), immunofluorescent staining of ICAM1 ( M , N ), immunofluorescent staining of CD68 + area ( O , P ), immunofluorescent staining of PDCD4 ( Q , R ), TUNEL staining apoptotic cells ( S , T ) of aortic root from the mice above ( n = 6 biological replicates). Scale bar: 50 μm. Statistical analyses were carried out via one-way ANOVA with the Bonferroni test. * P < 0.05, ** P < 0.01

Article Snippet: Membranes were blocked with 5% bovine serum albumin for 1 h at room temperature and incubated overnight at 4 °C with the following primary antibodies: CD9 (20597-1-AP, Proteintech, China), ALIX (12422-1-AP, Proteintech, China), CD63 (25682-1-AP, Proteintech, China), Calnexin (2679, CST, USA), Albumin (82783-6-RR, Proteintech, China), ApoA1 (14427-1-AP, Proteintech, China), VCAM1 (ab134047, Abcam, USA), ICAM1 (sc-8439, Santa Cruz, USA), BAX (50599-2-Ig, Proteintech, China), BCL2 (T40056S, Abmart, China), Caspase 3 (T40044S, Abmart, China), Cleaved Caspase 3 (TA7022S, Abmart, China), and GAPDH (2118 S, CST, USA).

Techniques: Derivative Assay, Fluorescence, Injection, Labeling, Ex Vivo, Imaging, Staining, In Vivo, TUNEL Assay

Journal: Journal of Nanobiotechnology

Article Title: COPD airway epithelial cells–derived extracellular vesicles contribute to endothelial dysfunction and atherosclerosis via the miR-141-3p/PDCD4 axis

doi: 10.1186/s12951-026-04091-0

Figure Lengend Snippet: EVs Derived from COPD Epithelial Cell Encapsulating agomiR-141-3p alleviate Atherosclerosis and Inhibit Vascular Inflammation and Apoptosis in ApoE -/-. ( A ) A scheme illustrating the preparation of agomiR-NC/141-3p loaded EVs. ( B ) NTA results showed the diameter of EVs. ( C ) Representative images of EVs morphology observed via TEM (Scale bar = 100 nm). ( D ) Representative western blot images of ALIX, CD9, CD63 and Calnexin. ( E ) Relative miR-141-3p levels in COPD EVs-agomiR NC/COPD EVs-agomiR-141-3p ( n = 3 biological replicates). ( F ) Experimental scheme: ApoE -/- Mice fed a high-fat diet and were injected with COPD EVs-agomiR NC or COPD EVs-agomiR-141-3p. ( G ) Representative en face Oil Red O–stained aortas and ( H ) corresponding quantification from ApoE -/- mice fed a high-fat diet and treated with COPD EVs-agomiR NC or COPD EVs-agomiR-141-3p ( n = 8 biological replicates). ( I ) Representative images of the thoracic aorta and carotid artery from the above groups after in vivo perfusion with 4% paraformaldehyde. White areas indicate arterial plaques. ( J-W ) Representative images and quantifications of Oil Red O staining ( J , K ), H&E staining (lesion area) ( L , M ), immunofluorescent staining of VCAM1 (N, O), immunofluorescent staining of ICAM1 ( P , Q ), immunofluorescent staining of CD68 + area ( R , S ), immunofluorescent staining of PDCD4 ( T , U ), TUNEL staining apoptotic cells ( V , W ) of aortic root from the mice above ( n = 8 biological replicates). Scale bar: 50 μm. P -values were calculated using Student’s t-test. * P < 0.05, ** P < 0.01

Article Snippet: Membranes were blocked with 5% bovine serum albumin for 1 h at room temperature and incubated overnight at 4 °C with the following primary antibodies: CD9 (20597-1-AP, Proteintech, China), ALIX (12422-1-AP, Proteintech, China), CD63 (25682-1-AP, Proteintech, China), Calnexin (2679, CST, USA), Albumin (82783-6-RR, Proteintech, China), ApoA1 (14427-1-AP, Proteintech, China), VCAM1 (ab134047, Abcam, USA), ICAM1 (sc-8439, Santa Cruz, USA), BAX (50599-2-Ig, Proteintech, China), BCL2 (T40056S, Abmart, China), Caspase 3 (T40044S, Abmart, China), Cleaved Caspase 3 (TA7022S, Abmart, China), and GAPDH (2118 S, CST, USA).

Techniques: Derivative Assay, Western Blot, Injection, Staining, In Vivo, TUNEL Assay