Fig. 1: Chromatin compaction and histone hypoacetylation in aged MSCs.

a, Scheme of the isolation protocol of bone marrow MSCs from the back limbs of young (~3–5 months old) and old (~18–22 months old) mice. b, Microarray (MA)-plot showing opening and closing peaks with age, as determined by ATAC-seq, averaged from n = 2 biologically independent experiments. c, Overall genome accessibility expressed as RPKM values measured by ATAC-seq. Statistical significance was determined by two-sided Wilcoxon test. The y axis is scaled to log₁₀ for visualization purposes. The lower and upper hinges of the boxplot correspond to the first and third quartiles (25th and 75th percentiles) while the middle line is median and the whiskers extend to 1.5 × interquartile range (IQR) from both lower and upper hinges. The notches extend 1.58 × IQR/sqrt(n), which is roughly 95% confidence intervals (CIs) for comparing medians; n = 2 of biologically independent experiments. d, Metaplot of ATAC-seq reads over the TSS of all protein-coding genes; n = 2 biologically independent experiments. e, NucleoATAC metaplot to map position of all nucleosomes around the TSS of all protein-coding genes. f, Representation of GO terms analyzed with Metascape⁶⁹ for opening and closing peaks upon aging, identified by ATAC-seq. GTPase-med. signal transd., GTPase-mediated signal transduction g,h, Representative images (g) and quantification (h) of mean fluorescence intensity (MFI), after labeling n = 140 young cells and n = 118 aged cells with 1 mM 5-EU, for 7 h. Nuclei were stained with DAPI. Merged results from n = 3 biologically independent samples are shown. i–l, Representative images (i,k) and quantification of mean fluorescence intensity (MFI) after immunostaining against H3ac and H3 of n = 203 young cells and n = 183 aged cells, and against H4ac and H4 of n = 115 young cells and n = 107 aged cells, respectively. MFI of histone H3 and histone H4 was used as internal control, respectively, for normalization. Nuclei were stained with DAPI; n = 3 biologically independent samples. Representative results from one replicate are shown in j and l. Scale bars (g, i and k), 25 μm. Distribution of data points in h, j and l is shown as violin plots, where the mean is indicated by a solid line and the quartiles are indicated with dashed lines. Statistical significance was determined in (h, j and l) using two-sided unpaired t-test.