Journal: bioRxiv

Article Title: Anti-inflammatory macrophages limit endothelial inflammation and restrict neutrophil recruitment through Gas6/AXL signaling

doi: 10.64898/2026.10.12.758565

Figure Lengend Snippet: (A) Schematic of the three-dimensional inflammation-on-a-chip device. A cylindrical lumen lined with human umbilical vein endothelial cells sits within a collagen–fibronectin extracellular matrix. Primary human monocyte-derived macrophages are polarized in situ within the matrix toward pro-inflammatory (Pro-) or anti-inflammatory (Anti-) phenotypes. Top and side ports allow injection of chemoattractants, neutrophils and inhibitors. (B) Cell viability (%) for pro- and anti-inflammatory macrophages on Days 6, 7 and 9. N = 3, n = 3. (C) Polarization Index (CD163 + /CD80 + ratio, log scale). Welch’s t-test on log 2 -transformed values, P = 0.007. N = 4–6, n = 3. (D) Anti/Pro cytokine secretion ratio (log 2 ) for pro- and anti-inflammatory macrophages in 3D collagen– fibronectin hydrogels, quantified by multiplex MagPix Luminex assay. The ratio is the summed concentrations of the three anti-inflammatory analytes (IL-10, soluble CD163 and MDC/CCL22) divided by the summed concentrations of the two pro-inflammatory analytes (IL-6 and TNF-α). Welch’s t-test on log 2 -transformed values, P = 0.0275. N = 4– 5, n = 1. (E) Representative confocal maximum-intensity projections of anti-inflammatory (top) and pro-inflammatory (bottom) macrophages in the 3D collagen–fibronectin matrix on Day 9, stained for Hoechst (nuclei, blue), CD68 (white), CD80 (red) and CD163 (green). Scale bar, 100 µm. In (B) to (D), the center line is the median, the box spans the interquartile range and the whiskers span the full rang(E) with individual biological replicates overlaid.

Article Snippet: Pooled primary human umbilical vein endothelial cells (HUVECs; PromoCell, C-12203 and 50305964) were maintained in Endothelial Growth Medium-2 (EGM-2; Lonza, NC9525043) in T-75 flasks between passages 2 and 7, with media refreshed every 2 days.

Techniques: Derivative Assay, In Situ, Injection, Transformation Assay, Multiplex Assay, Luminex, Confocal, Staining

Journal: bioRxiv

Article Title: Anti-inflammatory macrophages limit endothelial inflammation and restrict neutrophil recruitment through Gas6/AXL signaling

doi: 10.64898/2026.10.12.758565

Figure Lengend Snippet: (A) Schematic of the FITC-dextran permeability assay. A 70-kDa FITC-dextran solution is perfused into the HUVEC lumen and live-imaged at the vessel midplane by confocal microscopy, and the diffusive permeability coefficient Pd is computed as the slope of normalized extracellular matrix fluorescence accumulation over the 8-min window multiplied by the vessel-wall geometric factor . (B) Representative time-lapse confocal micrographs of FITC-dextran perfusion across the endothelial barrier at 2-min intervals (t = 0, 2, 4, 6 and 8 min). Red lines mark the vessel walls. Scale bar, 100 µm. (C) Condition mean of the normalized extracellular matrix fluorescence traces over the 8-min acquisition window. Solid lines, with stimulation. Dashed lines, without. (D) Diffusive permeability coefficient Pd (× 10 −6 cm/s). All six conditions were acquired within each experimental series, across independent biological replicates. Kruskal–Wallis test with Bonferroni-corrected pairwise post hoc comparisons. *P < 0.05. N = 7–9, n = 2–3. In (D), the center line is the median, the box spans the interquartile range and the whiskers span the full range, with individual biological replicates overlaid. (+) denotes inflammatory stimulation and (−) its absence.

Article Snippet: Pooled primary human umbilical vein endothelial cells (HUVECs; PromoCell, C-12203 and 50305964) were maintained in Endothelial Growth Medium-2 (EGM-2; Lonza, NC9525043) in T-75 flasks between passages 2 and 7, with media refreshed every 2 days.

Techniques: FITC-Dextran Permeability Assay, Confocal, Microscopy, Permeability, Fluorescence

Journal: eLife

Article Title: p66Shc mediates SUMO2-induced endothelial dysfunction

doi: 10.7554/eLife.106344

Figure Lengend Snippet: ( A ) Immunoblot for Shc and SUMO2 expression in HUVECs following p66Shc knockdown (si-p66Shc) and SUMO2 overexpression (Ad-HA-SUMO2). ( B, C ) Quantification of ROS (MitoSOX fluorescence) in HUVECs from A. *p<0.05; n=24–32; Student’s t -test. ( D ) Quantification of ROS in HUVECs following p66Shc knockdown (si-p66Shc) and SUMO2 overexpression (Ad-HA-SUMO2). ***p<0.001, p<0.05; n=10–15; One-way ANOVA. Data represents mean ± SEM. Immunoblots are representative of at least three independent experiments. GC, scrambled siRNA used as control; HUVECs, human umbilical vein endothelial cells. Figure 1—source data 1. PDF file containing original western blots for . Figure 1—source data 2. Original files for western blot analysis displayed in .

Article Snippet: Human umbilical vein endothelial cells (HUVECs) were purchased from PromoCell (Heidelberg, Germany) and Lonza (Walkersville, MD) and were cultured in endothelial growth medium-2 from PromoCell and Lonza.

Techniques: Western Blot, Expressing, Knockdown, Over Expression, Fluorescence, Control, Analysis

Journal: eLife

Article Title: p66Shc mediates SUMO2-induced endothelial dysfunction

doi: 10.7554/eLife.106344

Figure Lengend Snippet: ( A ) Immunoblot for sumoylated p66Shc in HEK-293 cells overexpressing SUMO2 (HA-SUMO2) and p66Shc (Flag-p66Shc). ( B ) Immunoblot for p66Shc sumoylation in HEK-293 cells overexpressing SUMO2 (HA-SUMO2) and p66Shc (Flag-p66Shc) with or without overexpression of Ubc9 (HA-Ubc9) and SENP1 (Flag-SENP1). ( C ) Immunoblot for sumoylation of p66Shc in human umbilical vein endothelial cells (HUVECs) overexpressing SUMO2 (Ad-HA-SUMO2) or p66Shc (Ad-Flag-p66Shc). ( D ) Immunoblot for p66Shc sumoylation in HUVEC cells overexpressing SUMO2 (Ad-HA-SUMO2) or p66Shc (Ad-Flag-p66Shc) in the presence or absence of anacardic acid. Quantification of SUMO2-p66Shc from immunoblot of D. One-way ANOVA, followed by Tukey’s post-hoc analysis, n=4 *p<0.05, **p<0.01. ( F ) Immunoblot for sumoylated recombinant p66Shc in the presence of absence of Ubc9. Immunoblots are representative of at least three independent experiments. Data represents mean ± SEM. Ad-Lac Z was used as an adenoviral control to compensate for the difference in MOI among the groups. Figure 2—source data 1. PDF file containing original western blots for . Figure 2—source data 2. Original files for western blot analysis displayed in .

Article Snippet: Human umbilical vein endothelial cells (HUVECs) were purchased from PromoCell (Heidelberg, Germany) and Lonza (Walkersville, MD) and were cultured in endothelial growth medium-2 from PromoCell and Lonza.

Techniques: Western Blot, Over Expression, Analysis, Recombinant, Control

Journal: eLife

Article Title: p66Shc mediates SUMO2-induced endothelial dysfunction

doi: 10.7554/eLife.106344

Figure Lengend Snippet: Immunoblot of human umbilical vein endothelial cells (HUVECs) transduced with wild-type p66Shc (Ad-Flag-p66ShcWT) and SUMO2 (Ad-HA-SUMO2) at the indicated concentrations. Immunoblots are representative of at least three independent experiments. Figure 2—figure supplement 1—source data 1. PDF file containing original western blots for . Figure 2—figure supplement 1—source data 2. Original files for western blot analysis displayed in .

Article Snippet: Human umbilical vein endothelial cells (HUVECs) were purchased from PromoCell (Heidelberg, Germany) and Lonza (Walkersville, MD) and were cultured in endothelial growth medium-2 from PromoCell and Lonza.

Techniques: Western Blot, Transduction, Analysis

Journal: eLife

Article Title: p66Shc mediates SUMO2-induced endothelial dysfunction

doi: 10.7554/eLife.106344

Figure Lengend Snippet: ( A ) Modular structure of p66Shc showing conserved lysine-81 in the CH2 domain. ( B ) Tandem mass spectrometry of in vitro SUMO2ylated recombinant p66Shc. ( C ) Immunoblot showing sumoylation of p66Shc in HEK-293 cells overexpressing p66ShcWT (Flag-p66ShcWT) or p66ShcK81R (Flag-p66ShcK81R) with and without SUMO2 (HA-SUMO2). ( D ) Immunoblot showing sumoylation of p66Shc in human umbilical vein endothelial cells (HUVECs) overexpressing p66ShcWT (Ad-Flag-p66ShcWT) or p66ShcK81R (Ad-p66ShcK81R) with and without SUMO2 (Ad-HA-SUMO2). ( E ) Immunoblot for sumoylation of recombinant p66ShcWT or p66ShcK81R. ( F ) Immunoprecipitation of SUMO2-p66Shc using SUMO2-p66ShcK81 antibody or the matching IgG control in HUVECs followed by immunoblotting with SUMO2-p66ShcK81 antibody showing endogenous SUMO2ylation of p66Shc (left-side panel, the high-intensity IgG signal was masked with a tape which appears as a white strip). Immunoblotting with Shc antibody in whole-cell lysate (right-side panel). Immunoblots are representative of at least three independent experiments. CH2, collagen homology 2; PTB, phosphotyrosine binding; CH1, collagen homology 1; SH2, Src homology 2. Figure 3—source data 1. PDF file containing original western blots for . Figure 3—source data 2. Original files for western blot analysis displayed in . Figure 3—source data 3. PDF file containing original western blots for . Figure 3—source data 4. Original files for western blot analysis displayed in . Figure 3—source data 5. PDF file containing original western blots for . Figure 3—source data 6. Original files for western blot analysis displayed in . Figure 3—source data 7. PDF file containing original western blots for . Figure 3—source data 8. Original files for western blot analysis displayed in .

Article Snippet: Human umbilical vein endothelial cells (HUVECs) were purchased from PromoCell (Heidelberg, Germany) and Lonza (Walkersville, MD) and were cultured in endothelial growth medium-2 from PromoCell and Lonza.

Techniques: Mass Spectrometry, In Vitro, Recombinant, Western Blot, Immunoprecipitation, Control, Stripping Membranes, Binding Assay, Analysis

Journal: eLife

Article Title: p66Shc mediates SUMO2-induced endothelial dysfunction

doi: 10.7554/eLife.106344

Figure Lengend Snippet: ( A ) Immunoblot for p66ShcS36 phosphorylation in HEK-293 cells overexpressing SUMO2 (HA-SUMO2) and p66ShcWT (Flag-p66ShcWT). ( B ) Quantification of p66ShcS36 phosphorylation from A. *p<0.05; n=6; one-way ANOVA. ( C ) Immunoblot for p66ShcS36 phosphorylation in human umbilical vein endothelial cells (HUVECs) overexpressing SUMO2 (Ad-HA-SUMO2) or p66ShcWT (Ad-Flag-p66ShcWT). ( D ) Quantification of p66ShcS36 phosphorylation from C. *p<0.05; n=8; one-way ANOVA. ( E ) Immunoblot for p66ShcS36 phosphorylation in HUVECs expressing SUMO2 (Ad-HA-SUMO2) and p66ShcWT (Ad-Flag-p66ShcWT) or p66ShcK81R (Ad-Flag-p66ShcK81R). ( F ) Quantification of p66ShcS36 phosphorylation from E. *p<0.05, **p<0.01; n=11; two-way ANOVA. Immunoblots were quantified with iBright image analysis software. Data represents mean ± SEM. Immunoblots are representative of at least three independent experiments. Figure 4—source data 1. PDF file containing original western blots for . Figure 4—source data 2. Original files for western blot analysis displayed in . Figure 4—source data 3. PDF file containing original western blots for . Figure 4—source data 4. Original files for western blot analysis displayed in . Figure 4—source data 5. PDF file containing original western blots for . Figure 4—source data 6. Original files for western blot analysis displayed in .

Article Snippet: Human umbilical vein endothelial cells (HUVECs) were purchased from PromoCell (Heidelberg, Germany) and Lonza (Walkersville, MD) and were cultured in endothelial growth medium-2 from PromoCell and Lonza.

Techniques: Western Blot, Phospho-proteomics, Expressing, Analysis, Software

Journal: eLife

Article Title: p66Shc mediates SUMO2-induced endothelial dysfunction

doi: 10.7554/eLife.106344

Figure Lengend Snippet: ( A ) Immunoblot for p66ShcWT in whole-cell lysates or the mitochondrial fraction of human umbilical vein endothelial cells (HUVECs) ectopically expressing SUMO2 (Ad-HA-SUMO2) and p66ShcWT (Ad-Flag-p66ShcWT). ( B ) Quantification of the relative abundance of p66Shc in the mitochondria from A. **p<0.01, ****p<0.0001; n=5; one-way ANOVA. ( C ) Immunoblot for p66Shc in whole-cell lysates or the mitochondrial fraction of HUVECs ectopically expressing SUMO2 (Ad-HA-SUMO2) and p66ShcWT Ad-Flag-p66ShcWT or p66ShcK81R (Ad-Flag-p66ShcK81R). ( D ) Quantification of relative abundance of p66Shc in the mitochondria from C. *p<0.05, **p<0.01; n=7; two-way ANOVA. Data represents mean ± SEM. Immunoblots are representative of at least three independent experiments. Figure 5—source data 1. PDF file containing original western blots for . Figure 5—source data 2. PDF file containing original western blots for . Figure 5—source data 3. PDF file containing original western blots for . Figure 5—source data 4. Original files for western blot analysis displayed in .

Article Snippet: Human umbilical vein endothelial cells (HUVECs) were purchased from PromoCell (Heidelberg, Germany) and Lonza (Walkersville, MD) and were cultured in endothelial growth medium-2 from PromoCell and Lonza.

Techniques: Western Blot, Expressing, Analysis

Journal: eLife

Article Title: p66Shc mediates SUMO2-induced endothelial dysfunction

doi: 10.7554/eLife.106344

Figure Lengend Snippet: ( A ) Immunoblot showing oxidized-LDL (o-LDL)-induced changes in small ubiquitin-like modifier 2/3 (SUMO2/3)-conjugated proteins in human umbilical vein endothelial cells (HUVECs) with and without SUMO2. ( B ) Graph showing serum cholesterol-level in wild-type, Ldlr -/- mice on a high fat diet, and Shc1 K81R mice on a high fat diet (n=4/group). One-way ANOVA, ****p<0.0001. Graphs showing % contraction following induction of endothelium-dependent relaxation ( C ) and endothelium-independent relaxation ( D ) in aortic rings from male wild-type mice, (n=14 rings from 5 mice), Ldlr -/- mice on a high fat diet (n=18 rings from 6 mice) and Ldlr - /- :: Shc1 K81R mice on a high fat diet (n=12 rings from 6 mice). Two-way ANOVA. ***p<0.001. ( E ) Representative images showing the level of 8-OHdG in aortic section of Ldlr -/- mice on a high fat diet (n=6 mice) and Ldlr -/- :: Shc1 K81R mice on a high fat diet (n=6 mice) and their ( F ) quantification. One-way ANOVA. *p<0.05. HFD, high fat diet; Ach, acetylcholine; SNP, sodium nitroprusside; PE, Phenylephrine; 8-OHdG, 8 hydroxy deoxyguanosine. Data represents mean ± SEM. Figure 7—source data 1. Source data for immunoblots in . Figure 7—source data 2. Original datasets for .

Article Snippet: Human umbilical vein endothelial cells (HUVECs) were purchased from PromoCell (Heidelberg, Germany) and Lonza (Walkersville, MD) and were cultured in endothelial growth medium-2 from PromoCell and Lonza.

Techniques: Western Blot, Ubiquitin Proteomics

Journal: eLife

Article Title: p66Shc mediates SUMO2-induced endothelial dysfunction

doi: 10.7554/eLife.106344

Figure Lengend Snippet: ( A ) Representative immunoblots of endothelial cell lysate expressing Flag-p66ShcWT, Flag-p66ShcK81R, and HA-SUMO2 (n=3 shown, n=6 performed). ( B ) Bubble chart representation of all the regulatory pathways impacted by SUMO2-p66Shc in endothelial cells, rescued by p66ShcK81R. Pathway enrichment analysis was performed using R-based Reactome pathway analysis and visualization packages on differential protein expression data obtained from HUVECs expressing SUMO2-p66ShcWT or SUMO2-p66ShcK81R compared with Ad-LacZ controls (n=6). Figure 8—figure supplement 1—source data 1. PDF file containing original western blots for . Figure 8—figure supplement 1—source data 2. Original files for western blot analysis displayed in .

Article Snippet: Human umbilical vein endothelial cells (HUVECs) were purchased from PromoCell (Heidelberg, Germany) and Lonza (Walkersville, MD) and were cultured in endothelial growth medium-2 from PromoCell and Lonza.

Techniques: Western Blot, Expressing, Analysis

Journal: eLife

Article Title: p66Shc mediates SUMO2-induced endothelial dysfunction

doi: 10.7554/eLife.106344

Figure Lengend Snippet: ( A, B ) Principal component analysis (PCA) of proteins expressed in endothelial cells expressing SUMO2+p66ShcWT and SUMO2+p66ShcK81R. ( C, D ) Bar chart presentation of key regulatory pathways differentially affected by small ubiquitin-like modifier 2 (SUMO2)-p66ShcWT ( C ) and SUMO2-p66ShcK81R ( D ) in endothelial cells. The protein expression data (fold change) were obtained from human umbilical vein endothelial cells (HUVECs) expressing SUMO2 with p66ShcWT or p66ShcK81R compared to those expressing Ad-Lac Z. Pathway enrichment analysis ( B ) was performed using R-based bioinformatics tools, including PROGENy/decoupleR pathway activity inference and visualization packages. Differentially regulated pathways associated with senescence, mitochondrial dysfunction, apoptosis signaling, and angiogenesis are shown. Data were derived from a sample size of n=6.

Article Snippet: Human umbilical vein endothelial cells (HUVECs) were purchased from PromoCell (Heidelberg, Germany) and Lonza (Walkersville, MD) and were cultured in endothelial growth medium-2 from PromoCell and Lonza.

Techniques: Analysis, Expressing, Ubiquitin Proteomics, Activity Assay, Derivative Assay

Journal: eLife

Article Title: p66Shc mediates SUMO2-induced endothelial dysfunction

doi: 10.7554/eLife.106344

Figure Lengend Snippet: Amplex Red assay performed in HUVECs with and without knockdown of p66Shc expressing SUMO2 (Ad-SUMO2) or a control virus (Ad-LacZ).

Article Snippet: Human umbilical vein endothelial cells (HUVECs) were purchased from PromoCell (Heidelberg, Germany) and Lonza (Walkersville, MD) and were cultured in endothelial growth medium-2 from PromoCell and Lonza.

Techniques: Amplex Red Assay, Knockdown, Expressing, Control, Virus