Journal: Advanced Science

Article Title: SIRT6 Lysine‐Demyristoylates ATF2 to Ameliorate Vascular Injury via PRKCD/VE‐Cadherin Pathway Regulating Vascular Endothelial Barrier

doi: 10.1002/advs.202504948

Figure Lengend Snippet: Exploration of lysine myristoylation in SIRT6 KO Cells. A,B) Investigate global myristoylation levels in SIRT6 knockout (KO) 293T cells, using western blot analysis to determine the effects of ALK14 treatment across a concentration gradient. C) A mass spectrum of peptides enriched by the H133Y mutant, highlighting the precision of the enrichment method. D) The conditions and results from MS analyses, providing data on the identification process. E) Features Gene Ontology (GO) enrichment analysis of identified myristoylated proteins, indicating their biological significance and functional categories. F) The subcellular localization of identified myristoylated proteins, offering insights into their roles within the cell. G) Depicts a protein interaction network centered on SIRT6, illustrating the connections and potential regulatory mechanisms involving myristoylated proteins. H) Analyzes identified myristoylation motifs using bioinformatics tools, contributing to the understanding of myristoylation patterns and preferences. I) A mass spectrum of ATF2 highlighting a specific myristoylation site, exemplifying the method's ability to pinpoint post‐translational modifications. J) Outlines the comprehensive approach of combining H133Y‐based immunoprecipitation with mass spectrometry (IP‐MS) for the identification of myristoylated proteins in cells treated with ALK14, elucidating the methodological framework and its application to studying protein myristoylation.

Article Snippet: Alkynyl myristic acid (Alk14) was bought from Click Chemistry Tools.

Techniques: Knock-Out, Western Blot, Concentration Assay, Mutagenesis, Functional Assay, Immunoprecipitation, Mass Spectrometry, Protein-Protein interactions

Journal: Advanced Science

Article Title: SIRT6 Lysine‐Demyristoylates ATF2 to Ameliorate Vascular Injury via PRKCD/VE‐Cadherin Pathway Regulating Vascular Endothelial Barrier

doi: 10.1002/advs.202504948

Figure Lengend Snippet: Biological function of myristoylation at K296 site of ATF2. A) Western blot analysis of ATF2 level in 293T under ALK14 treatment in a concentration gradient (0–20 µg mL −1 ) for 12 h (n = 3). B) Immunoprecipitation (IP) and CLICK IT assays were performed in the lysates of SIRT6 KO 293T to detect the myristoylation level of ATF2 (n = 3). Before lysis, cells were transfected with ATF2‐FLAG (WT) and ATF2 K296R‐FLAG (KR) plasmids treated with or without ALK14 (10 µg mL −1 ) for 12 h. C) IP and CLICK IT assays were performed in the lysates of SIRT6KO and WT cells to detect the myrsitoylation of ATF2 (n = 3). Before lysis, cells were transfected with ATF2‐FLAG (WT) and ATF2 K296R‐FLAG (KR) plasmids under ALK14 (10 µg mL −1 ) treatment for 12 h. D) Nucleoplasmic separation and western blot assays were performed in SIRT6 KD HMEC cells to detect the level of ATF2 in cytoplasm and nucleus. Before lysis, cells were transfected with ATF2 WT and KR plasmids under ALK14 (10 µg mL −1 ) treatment for 12 h or not. Immunofluorescence staining of ATF2 (red) in each group under ALK14 treatment or not. DAPI was used for counterstaining cellular nuclei (blue) (n = 10, scale bars = 20 µm). The histograms show the relative ATF2 level (%) in nuclear (n = 3). E) Nucleoplasmic separation and western blot assays were performed in SIRT6 KD HMEC cells to detect the level of ATF2 in cytoplasm and nucleus. Before lysis, cells were transfected with G60A, ATF2 WT and KR plasmids under ALK14 (10 µg mL −1 ) treatment for 12 h. Immunofluorescence staining of ATF2 (red) in each group under ALK14 treatment. DAPI was used for counterstaining cellular nuclei (blue) (n = 10, scale bars = 20 µm). The histograms show the relative ATF2 level (%) in nuclear (n = 3). F) Nucleoplasmic separation and western blot assays were performed in SIRT6 KD HMEC cells to detect the level of ATF2 in cytoplasm and nucleus. Before lysis, cells were transfected with G60A and ATF2 WT plasmids under ALK14 (10 µg mL −1 ) treatment for 12 h or not. Immunofluorescence staining of ATF2 (red) in each group under ALK14 treatment or not. DAPI was used for counterstaining cellular nuclei (blue) (n = 10, scale bars = 20 µm). The histograms show the relative ATF2 level (%) in nuclear (n = 3). Date is represented as means ± S.E.M. p value by two‐tailed t ‐test (A‐F).

Article Snippet: Alkynyl myristic acid (Alk14) was bought from Click Chemistry Tools.

Techniques: Western Blot, Concentration Assay, Immunoprecipitation, Lysis, Transfection, Immunofluorescence, Staining, Two Tailed Test

Journal: Advanced Science

Article Title: SIRT6 Lysine‐Demyristoylates ATF2 to Ameliorate Vascular Injury via PRKCD/VE‐Cadherin Pathway Regulating Vascular Endothelial Barrier

doi: 10.1002/advs.202504948

Figure Lengend Snippet: SIRT6 demyristoylated ATF2 to improve the endothelial permeability through PRKCD/VE‐Cadherin signaling pathway. A) Western blot analysis of VE‐Cadherin, FLAG, PRKCD level in HMEC‐1 transfected with ATF2 WT and KR plasmids treated with or without ALK14 (10 ug mL −1 ) for 12h. The histograms show the relative protein level (%) (n = 3). (B) Western blot analysis of VE‐Cadherin, PRKCD, SIRT6 level in HMEC‐1 transfected with G60A plasmids with or without ALK14 treatment. The histograms show the relative protein level (%) (n = 3). C) Western blot analysis of VE‐Cadherin, PRKCD, SIRT6 level in HMEC‐1 transfected with siPRKCD and G60A with ALK14 treatment. The histograms show the relative protein level (%) (n = 3). D) A brief schematic diagram of the role of siPRKCD in ATF2/PRKCD/VE‐Cadherin signaling pathway. E) A schematic diagram of the transwell permeability model. F) The histograms represented the quantitative assays of endothelial permeability in ATF2 WT and KR HMEC‐1 with or without ALK14 treatment (n = 3). G) The histograms represented the quantitative assay of endothelial permeability in HMEC‐1 transfected with G60A plasmids under ALK14 treatment (n = 3) or not. H) The histogram represented the quantitative assay of endothelial permeability of HMEC‐1 transfected with siPRKCD and G60A under ALK14 treatment (n = 3). Date is represented as means ± S.E.M. p value by two‐tailed t ‐test (A, B, C, E, F, G, H).

Article Snippet: Alkynyl myristic acid (Alk14) was bought from Click Chemistry Tools.

Techniques: Permeability, Western Blot, Transfection, Two Tailed Test

Journal: Advanced Science

Article Title: SIRT6 Lysine‐Demyristoylates ATF2 to Ameliorate Vascular Injury via PRKCD/VE‐Cadherin Pathway Regulating Vascular Endothelial Barrier

doi: 10.1002/advs.202504948

Figure Lengend Snippet: SIRT6 demyristoylated ATF2 to improve the endothelial permeability through PRKCD/VE‐Cadherin signaling pathway. A) Immunofluorescence staining analyses of VE‐Cadherin (green) in HMEC‐1 transfected with ATF2 WT and KR plasmids under ALK14 (10 µg mL −1 ) treatment for 12 h or not. Nuclei were counterstained with DAPI (blue) (n = 8–10, scale bars = 50 µm). The histograms show the distribution of the intercellular 5um or 10um gaps of each group (n = 8–10). B) Immunofluorescence staining analyses of VE‐Cadherin (green) in HMEC‐1 transfected with G60A plasmids under ALK14 treatment (n = 8–10, scale bars = 50 µm) or not. The histograms show the distribution of intercellular 5um or 10um gaps of each group (n = 8–10). C) Immunofluorescence staining analyses of VE‐Cadherin (green) in HMEC‐1 transfected with siPRKCD and G60A under ALK14 treatment (n = 8–10, scale bars = 50 µm). The histograms show the distribution of the intercellular 5 um or 10um gaps of each group (n = 8–10). Data were presented as mean ± S.E.M and analyzed using a two‐tailed t ‐test A,B,C). ns indicates no significant difference.

Article Snippet: Alkynyl myristic acid (Alk14) was bought from Click Chemistry Tools.

Techniques: Permeability, Immunofluorescence, Staining, Transfection, Two Tailed Test

Journal: Advanced Science

Article Title: SIRT6 Lysine‐Demyristoylates ATF2 to Ameliorate Vascular Injury via PRKCD/VE‐Cadherin Pathway Regulating Vascular Endothelial Barrier

doi: 10.1002/advs.202504948

Figure Lengend Snippet: ATF2/PRKCD/VE‐Cadherin signaling pathway was regulated by SIRT6 demyristoylase activity. A) Immunoprecipitation (IP) and CLICK IT assays were performed in the lysates of 293T SIRT6KO cells to detect the myristoylation level of ATF2 (n = 3). Before lysis, cells were transfected with SIRT6 mutants plasmids treated with ALK14 (10 µg mL −1 ) for 12 h. The histograms show the relative ATF2 myristoylation level (%) (n = 3). B) Western blot analysis of VE‐Cadherin, PRKCD level in HUVECs transfected with SIRT6 and its mutants plasmids treated with ALK14 (10 ug mL −1 ) for 12h. The histograms show the relative protein level (%) (n = 3). C) Immunofluorescence staining analyses of VE‐Cadherin (red) and ATF2(green) in HUVECs transfected with SIRT6 and its mutants treated with ALK14 (10 µg mL −1 ) for 12 h. The histograms (up) show the distribution of the intercellular 5 or 10 um gaps of each group. The histograms (down) show the ATF2 nuclear proportion. scar bar = 100 µm (n = 6–8) Data were presented as mean ± S.E.M and analyzed using a two‐tailed t ‐test. ns indicates no significant difference.

Article Snippet: Alkynyl myristic acid (Alk14) was bought from Click Chemistry Tools.

Techniques: Activity Assay, Immunoprecipitation, Lysis, Transfection, Western Blot, Immunofluorescence, Staining, Two Tailed Test