Journal: Cell Communication and Signaling : CCS

Article Title: α-Klotho prevents diabetic retinopathy by reversing the senescence of macrophages

doi: 10.1186/s12964-024-01838-w

Figure Lengend Snippet: Exploration of the mechanism of anti-senescence activity of KL. A Heatmap showing the DEPs in RAW264.7 cells with or without Ad-KL transfection. B Volcanic plot displaying the DEPs in RAW264.7 cells with or without Ad-KL transfection. C GO pathway analyses were employed to determine the biofunctions and associated signal paths for DEPs. D Top 10 E3 ligase substrates of HECTD1. E Heatmap showing the Top 10 DEGs in the GSE26168 datasets. F Venn diagrams showing common DEGs in DM and aging. G The PPI between 38 differentially expressed senescence-related genes was constructed using the STRING database. The node represents the gene, and the edge represents the gene relationship. H The first 10 genes identified through the maximal clique centrality method were screened as hub genes using the cytoHubba plugin. Increased red color represents more forward ranking

Article Snippet: HECTD1 knockout RAW264.7 cells were lysed and combined with 6 μg of anti-IRS1 (proteintech, Wuhan, China) per sample at 4 °C overnight.

Techniques: Activity Assay, Transfection, Construct

Journal: Cell Communication and Signaling : CCS

Article Title: α-Klotho prevents diabetic retinopathy by reversing the senescence of macrophages

doi: 10.1186/s12964-024-01838-w

Figure Lengend Snippet: KL alleviates macrophage senescence by downregulating HECTD1 to decrease IRS1 ubiquitination and degradation. A Molecular docking complexes of the HECTD1 solution NMR structure (green) and IRS1 PTB domain (blue) as obtained from ClusPro 2.0. The images were prepared using PyMOL. B , C RAW264.7 cells were transfected with HECTD1-siRNA for 72 h, followed by western blotting with anti-HECTD1 and anti-IRS1. The expression of HECTD1 and IRS1 in cells was investigated and quantified ( n = 3). D , E The expression of PI3K, p-AKT and AKT in cells was investigated and quantified ( n = 3). F RAW264.7 cells were transfected with HECTD1-siRNA for 72 h, followed by immunoprecipitation with anti-IRS1 and immunoblotting with anti-ubiquitin (anti-Ub). G - J RAW264.7 cells were further treated with 4HNE after transfection with ADV-GFP or ADV-KL. The expression of HECTD1 and IRS1 in cells was investigated and quantified ( n = 3). K , L The effects of KL on HECTD1 and IRS1 levels were observed by Western blot in the mouse PBMCs. Data represent the mean ± SEM. ∗ p < 0.05, ∗ ∗ p < 0.01, ∗ ∗ ∗ p < 0.001, by independent samples t-test and one-way ANOVA

Article Snippet: HECTD1 knockout RAW264.7 cells were lysed and combined with 6 μg of anti-IRS1 (proteintech, Wuhan, China) per sample at 4 °C overnight.

Techniques: Ubiquitin Proteomics, Transfection, Western Blot, Expressing, Immunoprecipitation