Journal: iScience

Article Title: Thrombospondin 1 aggravates cardiac remodeling in heart failure with preserved ejection fraction by inhibiting mitophagy

doi: 10.1016/j.isci.2026.114639

Figure Lengend Snippet: Thbs1 knockdown alleviates cardiomyocyte hypertrophy, oxidative stress, mitochondrial dysfunction, and mitophagy impairment in vitro (A) Schematic diagram of the in vitro pathological stimulation protocol. H9c2 cells were treated with isoproterenol (ISO) and macrophage-conditioned medium (MCM) derived from LPS+ATP-activated macrophages, with or without Thbs1 knockdown. (B and C) mRNA levels of hypertrophic markers ANP (B) and BNP (C) under pathological stimulation ( n = 3 biological replicates per group, each measured in triplicate). (D) Representative fluorescence images of mitochondrial reactive oxygen species (ROS) detected using MitoSOX Red. Scale bars, 20 μm. (E) Quantification of MitoSOX Red fluorescence intensity ( n = 6 biological replicates per group, each measured in triplicate). (F) JC-1 staining to assess mitochondrial membrane potential (MMP), representative images shown. Scale bars, 20 μm. (G) Quantification of JC-1 aggregates/monomers ratio ( n = 6 biological replicates per group, each measured in triplicate). (H) Immunofluorescence images showing LC3 (red) and VDAC (green) colocalization; nuclei were stained with DAPI (blue). Scale bars, 20 μm. (I) Representative western blot images of mitophagy-related proteins LC3, p62, PINK1, and Parkin. (J–M) Quantification of protein expression: LC3-II/I (J), p62 (K), PINK1 (L), and Parkin (M) ( n = 3 biological replicates per group, each measured in duplicate). Data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc test. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, indicating the level of statistical significance for each comparison.

Article Snippet: Subsequently, H9c2 cardiomyocytes were treated with MCM combined with isoproterenol (ISO; HY-B0468, MedChemExpress, Monmouth Junction, NJ, USA) for 24 h to mimic the combined inflammatory and adrenergic stress observed in HFpEF.

Techniques: Knockdown, In Vitro, Derivative Assay, Fluorescence, Staining, Membrane, Immunofluorescence, Western Blot, Expressing, Comparison

Journal: iScience

Article Title: Thrombospondin 1 aggravates cardiac remodeling in heart failure with preserved ejection fraction by inhibiting mitophagy

doi: 10.1016/j.isci.2026.114639

Figure Lengend Snippet: PI3K/Akt/mTOR pathway activation reverses the protective effects of Thbs1 knockdown in H9c2 cells (A) Representative western blot images showing phosphorylation and total levels of Akt and mTOR in H9c2 cells transfected with si- Thbs1 and treated with ISO plus MCM or the Akt activator SC79. (B and C) Quantification of protein phosphorylation: p -Akt/Akt (B) and p -mTOR/mTOR (C) ( n = 3 biological replicates per group, each measured in duplicate). (D and E) Relative mRNA expression of ANP (D) and BNP (E) assessed by RT-qPCR ( n = 3 biological replicates per group, each measured in triplicate). (F and G) Intracellular ROS production measured using MitoSOX Red mitochondrial superoxide indicator; representative fluorescence images are shown (F) and quantified as mean fluorescence intensity (G) ( n = 6 biological replicates per group, each measured in triplicate). Scale bars, 20 μm. (H) Representative confocal images of mitochondria-lysosome colocalization using Mito-Tracker (green) and Lyso-Tracker (red). Scale bars, 20 μm. (I) Representative western blot images of mitophagy-related proteins LC3, p62, PINK1, and Parkin. (J–M) Quantification of mitophagy-related protein expression: LC3-II/I (J), p62 (K), PINK1 (L), and Parkin (M) ( n = 3 biological replicates per group, each measured in duplicate). Data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc test. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, indicating the level of statistical significance for each comparison.

Article Snippet: Subsequently, H9c2 cardiomyocytes were treated with MCM combined with isoproterenol (ISO; HY-B0468, MedChemExpress, Monmouth Junction, NJ, USA) for 24 h to mimic the combined inflammatory and adrenergic stress observed in HFpEF.

Techniques: Activation Assay, Knockdown, Western Blot, Phospho-proteomics, Transfection, Expressing, Quantitative RT-PCR, Fluorescence, Comparison

Journal: iScience

Article Title: Thrombospondin 1 aggravates cardiac remodeling in heart failure with preserved ejection fraction by inhibiting mitophagy

doi: 10.1016/j.isci.2026.114639

Figure Lengend Snippet: Inhibition of PI3K/Akt/mTOR reverses effects of Thbs1 overexpression and Thbs1 binds ITGB1 in H9c2 cells (A) Representative western blot images showing phosphorylation and total levels of PI3K, Akt, and mTOR in H9c2 cells transfected with Thbs1 overexpression plasmid and treated with ISO plus MCM or the PI3K/Akt/mTOR inhibitor LY294002. (B–D) Quantification of protein phosphorylation: p-PI3K/PI3K (B), p -Akt/Akt (C), and p -mTOR/mTOR (D) ( n = 3 biological replicates per group, each measured in duplicate). (E and F) Intracellular ROS production measured using MitoSOX Red; representative fluorescence images are shown (E) and quantified as mean fluorescence intensity (F) ( n = 6 biological replicates per group, each measured in triplicate). Scale bars, 20 μm. (G) Representative confocal images showing mitochondria-lysosome colocalization using Mito-Tracker (green) and Lyso-Tracker (red). Scale bars, 20 μm. (H) Representative western blot images of mitophagy-related proteins LC3, p62, PINK1, and Parkin. (I–L) Quantification of mitophagy-related protein expression: LC3-II/I (I), p62 (J), PINK1 (K), and Parkin (L) ( n = 3 biological replicates per group, each measured in duplicate). (M) Co-immunoprecipitation (Co/IP) assays showing the interaction between Thbs1 and ITGB1 in H9c2 cells. Data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc test. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, indicating the level of statistical significance for each comparison.

Article Snippet: Subsequently, H9c2 cardiomyocytes were treated with MCM combined with isoproterenol (ISO; HY-B0468, MedChemExpress, Monmouth Junction, NJ, USA) for 24 h to mimic the combined inflammatory and adrenergic stress observed in HFpEF.

Techniques: Inhibition, Over Expression, Western Blot, Phospho-proteomics, Transfection, Plasmid Preparation, Fluorescence, Expressing, Immunoprecipitation, Co-Immunoprecipitation Assay, Comparison

Journal: iScience

Article Title: Thrombospondin 1 aggravates cardiac remodeling in heart failure with preserved ejection fraction by inhibiting mitophagy

doi: 10.1016/j.isci.2026.114639

Figure Lengend Snippet: Thbs1 knockdown alleviates cardiomyocyte hypertrophy, oxidative stress, mitochondrial dysfunction, and mitophagy impairment in vitro (A) Schematic diagram of the in vitro pathological stimulation protocol. H9c2 cells were treated with isoproterenol (ISO) and macrophage-conditioned medium (MCM) derived from LPS+ATP-activated macrophages, with or without Thbs1 knockdown. (B and C) mRNA levels of hypertrophic markers ANP (B) and BNP (C) under pathological stimulation ( n = 3 biological replicates per group, each measured in triplicate). (D) Representative fluorescence images of mitochondrial reactive oxygen species (ROS) detected using MitoSOX Red. Scale bars, 20 μm. (E) Quantification of MitoSOX Red fluorescence intensity ( n = 6 biological replicates per group, each measured in triplicate). (F) JC-1 staining to assess mitochondrial membrane potential (MMP), representative images shown. Scale bars, 20 μm. (G) Quantification of JC-1 aggregates/monomers ratio ( n = 6 biological replicates per group, each measured in triplicate). (H) Immunofluorescence images showing LC3 (red) and VDAC (green) colocalization; nuclei were stained with DAPI (blue). Scale bars, 20 μm. (I) Representative western blot images of mitophagy-related proteins LC3, p62, PINK1, and Parkin. (J–M) Quantification of protein expression: LC3-II/I (J), p62 (K), PINK1 (L), and Parkin (M) ( n = 3 biological replicates per group, each measured in duplicate). Data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc test. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, indicating the level of statistical significance for each comparison.

Article Snippet: Isoproterenol (ISO) , MedChemExpress , Cat# HY-B0468.

Techniques: Knockdown, In Vitro, Derivative Assay, Fluorescence, Staining, Membrane, Immunofluorescence, Western Blot, Expressing, Comparison