Journal: Oxidative Medicine and Cellular Longevity

Article Title: Acetylation of Atp5f1c Mediates Cardiomyocyte Senescence via Metabolic Dysfunction in Radiation-Induced Heart Damage

doi: 10.1155/2022/4155565

Figure Lengend Snippet: Ionizing radiation causes cardiomyocyte senescence. (a) Senescence-associated β -galactosidase (SA- β gal) staining of sham-irradiated and irradiated H9C2 cells. (b)–(d) Western blotting and quantification analyses of p21 and p16 protein expression in H9C2 cells in the control group and after 6h, 12h, and 24h of irradiation ( ∗ p < 0.05, ∗∗ p < 0.01). (e) q-PCR analysis of the mRNA levels of inflammation- and fibrosis-associated factors in H9C2 cells of the control, 6h, 12h, and 24h irradiation groups ( ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001). Bar: 100 μ m; h: hour.

Article Snippet: Total proteins from the animal heart apex or H9C2 cells were extracted using RIPA (Solarbio, Beijing, China), and protein concentration and western blotting were performed as previously described [ ] The primary antibodies used in this study were anti-acetyllysine rabbit pAb (1 : 1000; Jingjie PTM BioLabs, Inc., China), anti-Atp5f1 monoclonal antibody (1 : 1000; Proteintech, Wuhan, China), anti-p21 monoclonal antibody (1 : 1000; Boster, Wuhan, China), HA-tagged monoclonal antibody (1 : 20000; Proteintech, Wuhan, China), 6× His-tagged monoclonal antibody (1 : 10000; Proteintech, Wuhan, China), GAPDH monoclonal antibody (1 : 20000; Proteintech, Wuhan, China), and anti- α -tubulin monoclonal antibody (1 : 1000; Boster).

Techniques: Staining, Irradiation, Western Blot, Expressing, Control

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Acetylation of Atp5f1c Mediates Cardiomyocyte Senescence via Metabolic Dysfunction in Radiation-Induced Heart Damage

doi: 10.1155/2022/4155565

Figure Lengend Snippet: Atp5f1c is hyperacetylated at the 55th lysine site, which leads to metabolic disorders and senescence. (a) The expression of anti-His antibody after His-tagged Atp5f1c WT, K55R, and K55Q point mutation plasmids transfection in H9C2 cells. (b) Atp5f1c acetylation level verified by Co-IP after transfection of Atp5f1c WT, K55R, and K55Q point mutation plasmids in H9C2 cells. (c, d) Senescence-associated β -galactosidase (SABG) staining and statistical analysis after transfection of Atp5f1c WT, K55R, and K55Q point mutation plasmids in H9C2 cells ( ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; NS: not significant). (e) q-PCR analysis of the mRNA levels of inflammation- and fibrosis-associated factors after transfection of Atp5f1c WT, K55R, and K55Q point mutation plasmids in H9C2 cells ( ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001). (f)–(i) Western blotting and quantification analyses of p21 and p16 protein expression after transfection of Atp5f1c WT, K55R, and K55Q point mutation plasmids in H9C2 cells; relative protein levels were normalized to GAPDH or actin expression ( ∗ p < 0.05). (j, k) ATP synthase activity and ATP production after transfection of Atp5f1c WT, K55R, and K55Q plasmids into H9C2 cells ( ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; NS: not significant). Co-IP: Coimmunoprecipitation; WB: western blotting; WT: wild type.

Article Snippet: Total proteins from the animal heart apex or H9C2 cells were extracted using RIPA (Solarbio, Beijing, China), and protein concentration and western blotting were performed as previously described [ ] The primary antibodies used in this study were anti-acetyllysine rabbit pAb (1 : 1000; Jingjie PTM BioLabs, Inc., China), anti-Atp5f1 monoclonal antibody (1 : 1000; Proteintech, Wuhan, China), anti-p21 monoclonal antibody (1 : 1000; Boster, Wuhan, China), HA-tagged monoclonal antibody (1 : 20000; Proteintech, Wuhan, China), 6× His-tagged monoclonal antibody (1 : 10000; Proteintech, Wuhan, China), GAPDH monoclonal antibody (1 : 20000; Proteintech, Wuhan, China), and anti- α -tubulin monoclonal antibody (1 : 1000; Boster).

Techniques: Expressing, Mutagenesis, Transfection, Co-Immunoprecipitation Assay, Staining, Western Blot, Activity Assay