Supplementary Figure 2: Characterization of MSL3 patient-derived fibroblasts.

a, FFPE skin sections from Control (ctrl) and P1/P2/P14 stained with H&E. The dashed line distinguishes dermis and epidermis layers. Architectural skin layers are demarcated. Scale bar, 20 μm. Patients donated n = 1 skin sample; at least two sections per slide were analyzed. SB, stratum basale; SL, stratum lucidum; SS, stratum spinosum; SC, stratum corneum. b, Immunostaining for H3K27me3 (red) in primary HDFs. Scale bar, 5 µm. The staining was repeated twice with similar results. c, Cropped immunoblots for H4K16ac and total histone H3 as well as H3 and H4 pan-acetylation in additional HDF lines. The experiment was repeated twice with similar results. d, Sashimi plot derived from MiSeq results showing exon skipping in P1 but not P2 or Control (ctrl) HDF cDNA. e, Distribution of identified protein intensities measured in LC-MS/MS experiments before (left) and after (right) normalization. Protein intensities as well as modified site intensities were normalized and scaled by adjusting the centers of the distributions around zero to account for loading differences in SDS-PAGE. Box plots are centered on the median with the lower and upper hinges corresponding to the first and third quartiles. Normalized values were used for further statistical analysis as described in Supplementary Table 2. f, Heat map representing all acetyl (K), mono- and trimethyl (R-K) histone modification normalized intensities detected over the bulk histone background level as in Supplementary Table 2. g, Proliferation curve in P1, P2 and P14 compared to Control (ctrl). The center value at each time point represents the mean of n = 2 independent experiments. h, FACS cell cycle analysis of Control (ctrl) andP1/P2/P14 HDFs, Propidium iodide was used to define cell cycle phases. Bar plots represent the mean of n = 2 independent experiments with overlaid data points. i, RT–qPCR analysis of senescence markers P16-INK4A and P21-WAF displayed as dot plots. Expression levels were normalized to RPLP0 and expressed relative to Control (ctrl). Each data point represents an independent experiment (n) with the center line representing the mean ± s.e.m. when applicable. P values were determined by ordinary one-way ANOVA followed by Bonferroni multiple-test correction. Further details and statistical test values are provided in Supplementary Table 5. j, Representative DIC images of β-galactosidase activity assays performed in Control (ctrl) and P1/P2/P14 HDFs. The experiment was repeated three times with similar results. k, Representative FACS analysis of MKI67 (x axis) and H4K16ac (y axis) in Control (ctrl) and P1/P2/P14 HDFs. Quadrants show the percentage of cells with relative abundance of cell populations. The experiment was repeated twice with similar results.