Journal: iScience

Article Title: UFMylation maintains YAP stability to promote vascular endothelial cell senescence

doi: 10.1016/j.isci.2025.111854

Figure Lengend Snippet: YAP was upregulated in senescent endothelial cells (A) The proliferation of HUVECs population doubling level (PDL) at 10 and 30 were treated with vehicle or verteporfin (0.5 μM) for 12 h. Immunoblotting and quantifying the protein level of P16, P21, P53, VCAM1, and YAP. n = 4. (B) Proliferation of HUVECs PDL at 10 were transfected with siRNA-NC (negative control) or siRNA against YAP for 12 h, followed by treated with vehicle or H 2 O 2 (100 μM) for 12 h. Immunoblotting and quantifying the protein level of P16, P21, P53, VCAM1, and YAP. n = 4. (C) Proliferation of HUVECs PDL at 10 were infected with GFP adenovirus (Ad-GFP) or YAP adenovirus (Ad-YAP) for 24 h and then treated with vehicle or verteporfin (0.5 μM) for 12 h. Immunoblotting and quantifying the protein level of P16, P21, P53, VCAM1, and YAP. n = 4. (D and E) SA-β-Gal staining and quantitative analysis of senescent cells, scale bars: 40 μm, n = 6, p values correspond to one-way ANOVA with Tukey’s multiple comparisons test. All data are represented as mean ± SEM. ∗, p ≤ 0.05; ∗∗, p ≤ 0.01; ∗∗∗, p ≤ 0.001; ∗∗∗∗, p ≤ 0.0001.

Article Snippet: HUVEC (pooled donor) endothelial cells , Lonza , Cat# CC-2519.

Techniques: Western Blot, Transfection, Negative Control, Infection, Staining

Journal: iScience

Article Title: UFMylation maintains YAP stability to promote vascular endothelial cell senescence

doi: 10.1016/j.isci.2025.111854

Figure Lengend Snippet: YAP was identified as the substrate for UFMylation (A and B) Immunoblotting and quantification of the level of the indicated protein in HUVECs of the proliferation of PDL at 10 and 30; immunoblotting and quantification of levels of the indicated protein in aortic endothelial cells from 2 (young) or 24 (aged) months old mice. n = 3. (C) HEK293T cells were co-transfected with UFMylation components (UBA5, UFC1, UFL1, and DDRGK1) or wild-type (UFM1WT) or active UFM1 (UFM1ΔC2) or defective UFM1 (UFM1ΔC3) and FLAG-tagged YAP for detection exogenous YAP UFMylation. (D) Isolation of aortic endothelial cells from 8-week-old C57BL/6J mice for detection endogenous YAP UFMylation. (E) Immunofluorescence staining of YAP (green), UFM1 (red), and DAPI (blue) in HUVECs of the proliferation of PDL at 10 and 30, and serum stimulation as control, scale bars: 20 μm. (F) Isolated the nucleus and cytoplasmic proteins from young and senescent HUVECs, immunoblotting and quantification of levels YAP. n = 4. (G) UFMylation detection of YAP was identified using a PLA with UFM1 and YAP antibodies. n = 6, p values correspond to unpaired two-tailed Student’s t test. All data are represented as mean ± SEM. ∗, p ≤ 0.05; ∗∗, p ≤ 0.01; ∗∗∗, p ≤ 0.001; ∗∗∗∗, p ≤ 0.0001.

Article Snippet: HUVEC (pooled donor) endothelial cells , Lonza , Cat# CC-2519.

Techniques: Western Blot, Transfection, Isolation, Immunofluorescence, Staining, Control, Two Tailed Test

Journal: iScience

Article Title: UFMylation maintains YAP stability to promote vascular endothelial cell senescence

doi: 10.1016/j.isci.2025.111854

Figure Lengend Snippet: UFMylation of YAP slows down its protein degeneration speed and inhibiting UFMylation could prevent endothelial cell senescence (A and B) Proliferation of HUVECs PDL at 10 were transfected with siRNA-NC (negative control) or siRNA against UFL1/UFM1 for 36 h. Immunoblotting and quantifying the protein level of YAP, n = 4, p values correspond to one-way ANOVA with Tukey’s multiple comparisons test. (C and D) The proliferation of HUVECs PDL at 10 and 30 were treated with cycloheximide (CHX) (10 μM) for the indicated time. Immunoblotting and quantifying the protein level of YAP, n = 3, p values correspond to two-way ANOVA with Sidak’s multiple comparisons test. (E and F) Proliferation of HUVECs PDL at 30 were treated with vehicle or compound 8.5 (20 μM) for 12 h, followed by treated with CHX (10 μM) for the indicated time. Immunoblotting and quantifying the protein level of YAP, n = 3, p values correspond to two-way ANOVA with Sidak’s multiple comparisons test. (G and J) Proliferation of HUVECs PDL at 10 were treated with vehicle or H 2 O 2 (100 μM) for 12 h, followed by treated with vehicle, verteporfin (0.5 μM), compound 8.5 (20 μM) or verteporfin (0.5 μM), and compound 8.5 (20 μM) for 12 h. n = 3. (G and H) Immunoblotting and quantifying the indicated protein levels. (I and J) SA-β-Gal staining and quantitative analysis of senescent cells, scale bars: 40 μm, n = 6, p values correspond to one-way ANOVA with Tukey’s multiple comparisons test. All data are represented as mean ± SEM. ∗, p ≤ 0.05; ∗∗, p ≤ 0.01; ∗∗∗, p ≤ 0.001.

Article Snippet: HUVEC (pooled donor) endothelial cells , Lonza , Cat# CC-2519.

Techniques: Transfection, Negative Control, Western Blot, Staining

Journal: iScience

Article Title: UFMylation maintains YAP stability to promote vascular endothelial cell senescence

doi: 10.1016/j.isci.2025.111854

Figure Lengend Snippet:

Article Snippet: HUVEC (pooled donor) endothelial cells , Lonza , Cat# CC-2519.

Techniques: Control, Virus, Recombinant, Protease Inhibitor, Transfection, Bicinchoninic Acid Protein Assay, Western Blot, Staining, Negative Control, Software, Membrane