Journal: Biology

Article Title: Rapalink-1 Attenuates Oxidative-Stress-Induced Senescence in Vascular Cells in Association with Reduced NF-κB and MAPK Signaling

doi: 10.3390/biology15090732

Figure Lengend Snippet: ( A ) HUVECs were treated with increasing concentrations of H 2 O 2 (50–300 µM) for 24 h, and cell viability was assessed by MTT assay. ( B ) HUVECs were exposed to 300 µM H 2 O 2 in the presence of increasing concentrations of Rapalink-1 (50–400 pM) for 24 h, followed by viability measurement. Data are presented as mean ± SEM ( n = 3 independent experiments). Statistical significance was determined by one-way ANOVA with appropriate post hoc tests. Significance is indicated as: * p < 0.05, *** p < 0.001, **** p < 0.0001.

Article Snippet: Human umbilical-vein endothelial-cell (HUVEC) models were purchased from PromoCell (Heidelberg, Germany).

Techniques: MTT Assay

Journal: Biology

Article Title: Rapalink-1 Attenuates Oxidative-Stress-Induced Senescence in Vascular Cells in Association with Reduced NF-κB and MAPK Signaling

doi: 10.3390/biology15090732

Figure Lengend Snippet: ( A ) MTT assay results in HUVECs. ( B , C ) Representative images of DCFH-DA staining (green), Hoechst counterstaining (blue), merged images and quantification of DCFH-DA fluorescence intensity in HUVECs. ( D – F ) qPCR analysis in HUVECs: NRF2 , NOX4 , MnSOD . Scale bar = 50 μm. Data are presented as mean ± SD ( n = 3). Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test. Significance is indicated as: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Human umbilical-vein endothelial-cell (HUVEC) models were purchased from PromoCell (Heidelberg, Germany).

Techniques: MTT Assay, Staining, Fluorescence

Journal: Biology

Article Title: Rapalink-1 Attenuates Oxidative-Stress-Induced Senescence in Vascular Cells in Association with Reduced NF-κB and MAPK Signaling

doi: 10.3390/biology15090732

Figure Lengend Snippet: ( A , B ) Representative immunofluorescence images and quantification of γ-H2AX (green) in HUVECs with Hoechst nuclear counterstain (blue) and merged images. ( C , D ) Representative images and quantification of 8-OHDG (red) in HUVECs with Hoechst nuclear counterstain (blue) and merged images. Scale bar = 50 μm. Data are presented as mean ± SD ( n = 3). Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test. Significance is indicated as: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Human umbilical-vein endothelial-cell (HUVEC) models were purchased from PromoCell (Heidelberg, Germany).

Techniques: Immunofluorescence

Journal: Biology

Article Title: Rapalink-1 Attenuates Oxidative-Stress-Induced Senescence in Vascular Cells in Association with Reduced NF-κB and MAPK Signaling

doi: 10.3390/biology15090732

Figure Lengend Snippet: ( A , B ) Representative images and quantification of SA-β-gal staining in HUVECs. ( C , D ) Representative immunofluorescence images and quantification of Lamin B1 (red) in HUVECs with Hoechst counterstain (blue) and merged images. ( E , F ) Representative Western blots and quantification of the senescence-associated cell cycle regulator p21 in HUVECs. ( G ) qPCR analysis of p16 mRNA expression in HUVECs. Protein expression was normalized to GAPDH. Scale bar = 50 μm. Data are presented as mean ± SD ( n = 3). Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test. Significance is indicated as: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Human umbilical-vein endothelial-cell (HUVEC) models were purchased from PromoCell (Heidelberg, Germany).

Techniques: Staining, Immunofluorescence, Western Blot, Expressing

Journal: Biology

Article Title: Rapalink-1 Attenuates Oxidative-Stress-Induced Senescence in Vascular Cells in Association with Reduced NF-κB and MAPK Signaling

doi: 10.3390/biology15090732

Figure Lengend Snippet: ( A – F ) qPCR analysis of SASP-related inflammatory, cytokine, adhesion-related, and matrix-remodeling factors in HUVECs, including COX2 , IL-6 , IL-8 , ICAM1 , MMP1 , and TIMP1 . ( G – I ) Representative Western blots and quantification of relative protein expression of MMP2 and VCAM1 in HUVECs. Protein expression was normalized to GAPDH. Data are presented as mean ± SD ( n = 3). Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test. Significance is indicated as: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Human umbilical-vein endothelial-cell (HUVEC) models were purchased from PromoCell (Heidelberg, Germany).

Techniques: Western Blot, Expressing

Journal: Biology

Article Title: Rapalink-1 Attenuates Oxidative-Stress-Induced Senescence in Vascular Cells in Association with Reduced NF-κB and MAPK Signaling

doi: 10.3390/biology15090732

Figure Lengend Snippet: ( A ) Representative Western blot images showing the protein levels of p65, p-p65, p38, p-p38, ERK, and p-ERK (HUVECs). ( B – D ) Quantitative analysis of p-p65/p65, p-p38/p38, and p-ERK/ERK (HUVECs). Protein expression was normalized to GAPDH. Data are presented as mean ± SD ( n = 3). Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test. Significance is indicated as: ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Human umbilical-vein endothelial-cell (HUVEC) models were purchased from PromoCell (Heidelberg, Germany).

Techniques: Western Blot, Expressing

Journal: Biology

Article Title: Rapalink-1 Attenuates Oxidative-Stress-Induced Senescence in Vascular Cells in Association with Reduced NF-κB and MAPK Signaling

doi: 10.3390/biology15090732

Figure Lengend Snippet: ( A ) Representative Western blot images showing the protein levels of mTOR, p-mTOR, p-S6, p-4EBP1 (HUVECs). ( B – E ) Quantitative analysis of p-mTOR/mTOR, p-S6, p-4EBP1, p-AKT (HUVECs). Protein expression was normalized to GAPDH. Data are presented as mean ± SD ( n = 3). Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test. Significance is indicated as: ** p < 0.01, *** p < 0.001.

Article Snippet: Human umbilical-vein endothelial-cell (HUVEC) models were purchased from PromoCell (Heidelberg, Germany).

Techniques: Western Blot, Expressing

Journal: Cell Reports Medicine

Article Title: Enalaprilat reverses neutrophil polarization imbalance via targeting taurine-STING axis for treatment of diabetic wounds

doi: 10.1016/j.xcrm.2026.102714

Figure Lengend Snippet: Phenotype repolarization of neutrophils induced by Ena affected the pro-angiogenic capacities of HUVECs and inflammatory state of macrophages (A) Flow cytometry was adopted to reveal the difference of CD206+ population ratio among the three groups. n = 3 independent experiments. (B) Effects of AGE stimulation and sequential Ena supplement on the expression of ICAM1, CXCR2, CXCR4, and Fas as evaluated by western blot. (C) Effects of AGE stimulation and sequential Ena supplement on the expression of ICAM1, CXCR2, CXCR4, and Fas as evaluated by immunofluorescence staining. Scale bar, 2 μm; n = 3 independent experiments. (D) Quantitative reverse-transcription PCR (RT-qPCR) was employed to quantify the level of il1b , ccl3 , arg1 , and ccl17 . n = 3 independent experiments. (E) Schematic diagram for the incubation of HUVECs with pretreated neutrophils. (F) Proliferative ability of HUVECs under the stimulation of neutrophils with indicated preconditioning as detected via EdU staining. Scale bar, 200 μm; n = 3 independent experiments. (G) Migration property of HUVECs as measured by transwell assay. Scale bar, 200 μm; n = 3 independent experiments. (H) Migration property of HUVECs as measured by wound scratch test. Scale bar, 500 μm. (I) Tube formation assay was performed to visualize the neovascularization function of HUVECs with different treatments. Scale bar, 200 μm; n = 3 independent experiments. (J) Phenotype characters of macrophages irritated by neutrophils as determined by immunofluorescence staining. Scale bar, 5 μm. (K) Phenotype characters of macrophages irritated by neutrophils as determined by flow cytometry. n = 3 independent experiments. Data were shown as mean ± standard deviation (SD) from biological replicates, and statistical analyses were performed using one-way ANOVA test followed by Tukey’s multiple comparisons test in (A–D and F–K). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: Neutrophil cell line (HL-60) and human umbilical vein endothelial cell (HUVEC) were gained from the American Type Culture Collection (ATCC) and grown at 37°C with 5% CO 2 .

Techniques: Flow Cytometry, Expressing, Western Blot, Immunofluorescence, Staining, Reverse Transcription, Quantitative RT-PCR, Incubation, Migration, Transwell Assay, Tube Formation Assay, Standard Deviation

Journal: Cell Reports Medicine

Article Title: Enalaprilat reverses neutrophil polarization imbalance via targeting taurine-STING axis for treatment of diabetic wounds

doi: 10.1016/j.xcrm.2026.102714

Figure Lengend Snippet: Inhibitory effects of Ena on HUVEC ferroptosis activated by AGE-elicited neutrophils (A) CCK-8 assay was performed to measure the viability of HUVECs. n = 3 independent experiments. (B) Death rate of HUVECs incubated with neutrophils pretreated by different strategies as detected using flow cytometry. n = 3 independent experiments. (C) Levels of MDA and GSH were quantified to assess the ferroptosis activity. n = 3 independent experiments. (D) Intracellular lipid peroxidation was visualized by fluorescence staining. Scale bar, 10 μm; n = 3 independent experiments. (E) Fe 2+ ion content was visualized by fluorescence staining. Scale bar, 10 μm; n = 3 independent experiments. (F) JC-1 kit was used to evaluate the mitochondrial membrane potential of HUVECs. Scale bar, 10 μm; n = 3 independent experiments. (G) DCFH-DA fluorescence probe was applied to determine ROS abundance in HUVECs. Scale bar, 200 μm; n = 3 independent experiments. (H) Mitochondrial morphology in HUVECs as analyzed by transmission electron microscopy. Scale bar, 500 nm; n = 3 independent experiments. (I) Western blot detection of PGC1α and KLF9 in HUVECs with different treatments. n = 3 independent experiments. (J) Expression of KLF9 in HUVECs incubated with neutrophils pretreated by different approaches as detected using immunofluorescence staining. Scale bar, 10 μm; n = 3 independent experiments. (K) Expression of PGC1α in HUVECs incubated with neutrophils pretreated by different approaches as detected using immunofluorescence staining. Scale bar, 10 μm; n = 3 independent experiments. (L) Schematic diagram of Ena-elicited alleviation on endothelial cell ferroptosis induced by pro-inflammatory neutrophils. Data were shown as mean ± standard deviation (SD) from biological replicates, and statistical comparisons were performed using one-way ANOVA followed by Tukey’s multiple comparisons test in (A–G and I–K). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: Neutrophil cell line (HL-60) and human umbilical vein endothelial cell (HUVEC) were gained from the American Type Culture Collection (ATCC) and grown at 37°C with 5% CO 2 .

Techniques: CCK-8 Assay, Incubation, Flow Cytometry, Activity Assay, Fluorescence, Staining, Membrane, Transmission Assay, Electron Microscopy, Western Blot, Expressing, Immunofluorescence, Standard Deviation