bleomycin hydrochloride  (MedChemExpress)

Journal: Nature Communications

Article Title: Sub1 contributes to heart failure with preserved ejection fraction driven by aging in mice

doi: 10.1038/s41467-026-75237-9

Figure Lengend Snippet: a Identification of Sub1 as a novel anti-aging target in HFpEF. Differentially expressed genes (DEGs) in the E-MTAB-7454 dataset ( n = 11 controls vs. 5 HFpEF) were overlapped with DEGs in the PRJEB62450 dataset ( n = 4 controls vs. 6 HFpEF). b Sub1 expression levels in heart samples from non-HFpEF donors and HFpEF patients ( n = 11 vs. 5 and n = 4 vs. 6, respectively). c Single-cell RNA sequencing analysis of Sub1 expression in cardiomyocytes from different age groups: middle-aged (20–59 years) and old (60–100 years) ( n = 32567 and 36854 cells per group). The box represents the interquartile range (IQR), the center line indicates the median, and whiskers represent minimum and maximum values. d Sub1 expression in donor heart tissues from the GTEx database across the age range of 20–80 years ( n = 14, 147, 154, 66, 26, and 22 per group). e Correlation analysis between Sub1 and aging-associated genes from the GenAge database using RNA-seq data of human heart samples from GTEx. The top 36 correlated genes are shown. f Sub1 mRNA levels in HL-1 cells treated with bleomycin for 6, 12, 24, 36, or 48 h (( n = 4 biological replicates). g Representative western blots of Sub1, the DNA damage marker γ-H2AX, and cell senescence markers (p53, p21, and p16) in HL-1 cells treated with bleomycin at the indicated time points (( n = 4 per group). h Representative western blots of Sub1 and cell senescence markers (p53, p21, p16) in mouse heart tissues at the indicated ages (( n = 4 biological replicates). For ( b , c ), P -values were determined by two-tailed unpaired Student’s t-test. For ( d , f ), p values were determined by one-way ANOVA followed by Bonferroni’s post hoc test. Data are presented as mean ± SD.

Article Snippet: We used 50 μg/ml bleomycin (MCE, HY-17565A) to induce the HL-1 for 24 h to construct a cardiomyocyte senescence model . MG-132 (2 μM, MCE, HY-13259) was applied for 24 h to inhibit proteasome activity.

Techniques: Expressing, Single Cell, RNA Sequencing, Western Blot, Marker, Two Tailed Test

Journal: Nature Communications

Article Title: Sub1 contributes to heart failure with preserved ejection fraction driven by aging in mice

doi: 10.1038/s41467-026-75237-9

Figure Lengend Snippet: a Schematic of quantitative proteomic screening to identify Sub1-binding proteins in a cellular senescence model. b Table of Sub1-interacting proteins significantly altered by bleomycin treatment, ranked by interaction ratio. c Co-immunoprecipitation (Co-IP) validating interactions between Sub1 and TAF9b or p53 in HEK293T cells ( n = 3 independent experiments). d mRNA levels of p53 in Sub1-overexpressing and Sub1-knockdown HL-1 cells treated with bleomycin for 24 h ( n = 4 per group). e , f Representative western blots and quantification of p53 protein turnover in Sub1-overexpressing and Sub1-knockdown HL-1 cells treated with cycloheximide (CHX, 10 μM) and bleomycin ( n = 3 independent experiments). g Representative western blots of p53 in Sub1-overexpressing and Sub1-knockdown HL-1 cells treated with proteasome inhibitor MG132 (20 μM) and bleomycin ( n = 3 independent experiments). h p53 immunoprecipitation showing ubiquitinated p53 levels in bleomycin-treated Sub1-overexpressing and Sub1-knockdown HL-1 cells ( n = 3 independent experiments). i Schematic of the structural domains and interactions among Sub1, TAF9b, and p53. j , k Co-IP assays mapping the binding regions of TAF9b with Sub1 and Sub1 with TAF9b in HEK293T cells ( n = 3 independent experiments). l Representative western blots of Sub1, TAF9b, p53, p21, and p16 in Sub1-overexpressing HL-1 cells treated with bleomycin and si-TAF9b ( n = 3 independent experiments). m Representative SPiDER β-gal staining of Sub1-overexpressing HL-1 cells treated with bleomycin and si-TAF9b. Scale bars, 20 μm ( n = 6 per group). n p53 immunoprecipitation showing ubiquitinated p53 levels in Sub1-overexpressing HL-1 cells treated with bleomycin and si-TAF9b ( n = 3 independent experiments). For ( d ), p -values were determined by one-way ANOVA followed by Bonferroni’s post hoc test. Data are presented as mean ± SD.

Article Snippet: We used 50 μg/ml bleomycin (MCE, HY-17565A) to induce the HL-1 for 24 h to construct a cardiomyocyte senescence model . MG-132 (2 μM, MCE, HY-13259) was applied for 24 h to inhibit proteasome activity.

Techniques: Binding Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Knockdown, Western Blot, Staining

Journal: Nature Communications

Article Title: Sub1 contributes to heart failure with preserved ejection fraction driven by aging in mice

doi: 10.1038/s41467-026-75237-9

Figure Lengend Snippet: a Co-IP assays validating the interaction between exogenous Sub1 and AROS in HEK293T cells ( n = 3 independent experiments). b Representative western blots of Sub1 and acetylated p53 (K382) in Sub1-overexpressing and Sub1-knockdown HL-1 cells treated with bleomycin ( n = 3 independent experiments). c Representative western blot of Sub1 and acetylated p53 (K382) in aged Ctrl fl/fl, Sub1 Tg and Sub1 + /+, Sub1 −/+ mice, ( n = 3 independent experiments). d Representative western blots of acetylated p53 (K382) and SIRT1 in HL-1 cells transfected with increasing amounts of Flag-Sub1 plasmid ( n = 3 independent experiments). e Schematic of the structural domains and interactions among Sub1, AROS, and SIRT1. f Co-IP mapping the binding regions of AROS with Sub1 and SIRT1 in HEK293T cells ( n = 3 independent experiments). g Co-IP of SIRT1 in HEK293T cells co-transfected with Flag-Sub1 and HA-AROS plasmids (n = 3 independent experiments). h Representative SPiDER β-gal staining of Sub1-overexpressing HL-1 cells treated with bleomycin and HA-AROS. Scale bars, 20μm (n = 6 per group). i Representative western blots of Sub1, HA, acetylated p53 (K382), total p53, p21, and p16 in Sub1-overexpressing HL-1 cells treated with bleomycin and HA-AROS ( n = 3 independent experiments). j Representative western blots of Sub1, p53, p21, and p16 in Sub1-overexpressing HL-1 cells treated with bleomycin, Mutpep, and Acpep at the indicated concentrations ( n = 3 independent experiments). k Quantification of p53 transcriptional activity on Cdkn1a (p21) promoter using luciferase assays in Sub1-overexpressing HL-1 cells treated with bleomycin, Mutpep, and Acpep, ( n = 3 independent experiments). l Representative immunofluorescence (IF) images of cardiac organoids treated with bleomycin and indicated peptide, ( n = 6 per group). For ( k ), p -values were determined by one-way ANOVA followed by Bonferroni’s post hoc test. Data are presented as mean ± SD.

Article Snippet: We used 50 μg/ml bleomycin (MCE, HY-17565A) to induce the HL-1 for 24 h to construct a cardiomyocyte senescence model . MG-132 (2 μM, MCE, HY-13259) was applied for 24 h to inhibit proteasome activity.

Techniques: Co-Immunoprecipitation Assay, Western Blot, Knockdown, Transfection, Plasmid Preparation, Binding Assay, Staining, Activity Assay, Luciferase, Immunofluorescence