Schematic representation depicting experimental design and the incubation duration used in these experiments. Sphinganine induces DNA damage in human cardiomyocytes depicted here by γH2A.X+ single nuclei.
Micrographs depicting DHS‐induced DNA damage in human cardiomyocytes depicted here by γH2A.X+ nuclei in cardiac‐specific ACTN2+ cells (n = 3 biological replicates; no. of cells quantified = 147 per condition). ACTN2 is to specifically label human cardiomyocytes.
Bar graph depicting the number of γH2A.X+ nuclei in the indicated conditions.
Sphinganine induces DNA damage in human cardiomyocytes depicted here by neutral comet assay of DHS‐treated hCMs.
Quantification of comet length in the indicated conditions. Data are represented as box‐plot, and the extremes of the error bars represent non‐outlier range and their length represents the variability within the data. Horizontal line within the bars represents median of the underlying population. Box plot whiskers show 1.5 IQR of highest and lowest quartile, outliers are included (dots). Extremes of the error bars represent non‐outlier range and their length represents the variability within the data. Horizontal line and white dot within the bars represent median and mean of the underlying population, respectively.
Elevated DHS levels lead to ageing signatures in hCMs shown here by SA‐β‐GAL activity.
Quantification of SA‐β‐GAL activity assay in the indicated conditions. (n = 3 biological replicates). Data are represented as bar graph depicting percentage mean of the SA‐β‐GAL labelled area.
Elevated DHS levels lead to ageing signatures in hCMs shown here by p21 activation. Respective graphs depict the quantification of p21‐positive nuclei is on the right. (n = 3 biological replicates; number of cells quantified per replicate = 150–200).
Elevated DHS levels lead to ageing signatures in hCMs shown here by HP1‐α+ nuclear foci. Respective graphs depict the quantification of HP1‐α‐positive nuclei is on the right. (n = 3 biological replicates; number of cells quantified per replicate = 120–200).
Elevated DHS levels lead to ageing signatures in hCMs shown here by disruption of the nuclear envelope marked by Lamin B1 along with an increase in H4K16ac staining (n = 3 biological replicates; number of cells quantified per replicate = 100–150). Respective graphs depict the quantification of nuclear membrane defect is on the right.
Proteome analysis confirms sphinganine‐induced disruption of (epi)genomic integrity (n = 3). Noteworthy, comparison of the proteomic profiles was performed between hCMs pretreated with DMSO and DHS.
High sphinganine levels cause significant increase in H3K27ac and H3K56ac marks in hCMs. ACTN2 is used to specifically label human cardiomyocytes.
Ageing leads to significant increase in H3K27ac and H3K56ac marks in human hearts. Insets on the right depict magnified view of cardiomyocyte nuclei. ACTN2 is used to specifically label human cardiomyocytes.
Violin plot depicting the distributions of the greyscale nuclear intensity of the indicated markers. Quantification represents measurements of ˜ 150 single nuclei per condition, derived from three biological replicates.
Data information: ACTN2 is used to specifically label human cardiomyocytes. When not specified, the experiments were conducted in at least three biological replicates. Error bars in panels (C, H, I and J) represent standard error of the mean. For pairwise comparisons, Student's
t‐test was performed for the estimation of the statistical significance. For the comparison of fluorescence signal intensities (panel N), KS‐test was used as a measure of statistical significance.
P‐value cut‐off used for computing statistical significance is < 0.05. *, ** and *** in the figure refer to
P‐values ≤ 0.05, ≤ 0.01 and ≤ 0.001, respectively. Statistically non‐significant comparisons are annotated as ns. Scale bar = 50 μm (panel A), 10 μm (panels B, F, H, I, J and L) and 10 μm (panel M) and for insets 2 μm.
