Journal: iScience

Article Title: Topical application of urolithin A activates mitophagy and enhances mitochondrial function to promote skin longevity

doi: 10.1016/j.isci.2026.117646

Figure Lengend Snippet:

Article Snippet: The skin cells used in the study included Normal Human Dermal Fibroblasts (NHDF) sourced from two donors purchased from Promocell, (Catalog C12302 , age 26) and (Catalog C12302 , age 69).

Techniques: Binding Assay, Ex Vivo, Recombinant, Metals, Enzyme-linked Immunosorbent Assay, Reverse Transcription, Cell Viability Assay, Staining, DC Protein Assay, Gene Expression, Glass, Software, Imaging, Microscopy, Real-time Polymerase Chain Reaction

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

Journal: iScience

Article Title: Topical application of urolithin A activates mitophagy and enhances mitochondrial function to promote skin longevity

doi: 10.1016/j.isci.2026.117646

Figure Lengend Snippet: UA beneficial impact on human keratinocytes and reconstituted human epidermis (A) Mitochondrial respiration (basal and maximal) assessed as oxygen consumption rate (OCR) in human dermal keratinocytes after 72 h of differentiation from young donor treated with DMSO for 24 h or the indicated dose of UA. ∗ p < 0.05 and ∗∗∗ p < 0.001 after one-way ANOVA. Error bars represent mean ± SEM. (B) TOMM20 immunostaining of skin epidermis in a human full-thickness skin model from an old donor treated with or without UA 1 μM for 25 days. Data expressed as antibody staining signal over imaging field. ∗ p < 0.05 and ∗∗∗∗ p < 0.0001 after unpaired t test one-way ANOVA. Error bars represent mean ± SEM. Scale bars, 50 μm. (C) Relative gene expression of PINK1 and PARK2 in reconstructed human epidermis (RHE). RHE tissues were moisturized for 24 h with or without 1% Mitopure, and relative expression levels of PINK1 and PARK2 were quantified. Statistical analysis was performed using one-way ANOVA (∗ p < 0.05; ∗∗ p < 0.01). Error bars represent mean ± SEM. (D) Interleukin 6 levels normalized to cell viability in keratinocytes. Keratinocytes were stimulated with a pro-inflammatory cytokine mix for 24 h then treated for an additional 24 h with either DMSO or 1 μM UA. IL-6 secretion was quantified and normalized to cell viability. Statistical analysis was performed using one-way ANOVA (∗∗∗∗ p < 0.0001). Error bars represent mean ± SEM. (E) p16 immunostaining in a human full-thickness skin model from an old donor treated with or without UA 1 μM for 25 days. Data expressed as antibody staining signal over imaging field. ∗∗∗∗ p < 0.0001 after unpaired t test. Error bars represent mean ± SEM. Scale bars, 50 μm. (F) Ki67 immunostaining in a human full-thickness skin model from an old donor treated with or without UA 1 μM for 25 days. Data expressed as number of Ki67 positive cells normalized over μm of dermal epidermal junction (count/μm) ∗∗∗∗ p < 0.0001 after unpaired t test. Error bars represent mean ± SEM. Scale bars, 50 μm. (G) Keratin 10 immunostaining in a human full-thickness skin model from an old donor treated with or without UA 1 μM for 25 days. Data expressed as fluorescence intensity over epidermis area. ∗∗∗∗ p < 0.0001 after unpaired t test. Error bars represent mean ± SEM. Scale bars, 50 μm.

Article Snippet: Human: Normal Human Epidermal Keratinocytes (NHEK) , PromoCell , Cat#C-12003; Lot#479Z011.1.

Techniques: Immunostaining, Staining, Imaging, Gene Expression, Expressing, Analysis, Fluorescence

Journal: iScience

Article Title: Topical application of urolithin A activates mitophagy and enhances mitochondrial function to promote skin longevity

doi: 10.1016/j.isci.2026.117646

Figure Lengend Snippet:

Article Snippet: Human: Normal Human Epidermal Keratinocytes (NHEK) , PromoCell , Cat#C-12003; Lot#479Z011.1.

Techniques: Binding Assay, Ex Vivo, Recombinant, Metals, Enzyme-linked Immunosorbent Assay, Reverse Transcription, Cell Viability Assay, Staining, DC Protein Assay, Gene Expression, Glass, Software, Imaging, Microscopy, Real-time Polymerase Chain Reaction