Suv39h DKO lymphocytes show molecular alterations consistent with an aged phenotype. (A) Western blot analysis of H3K9me3 levels from CD45.2⁺ DP thymocytes (50 000 cells loaded per lane) of different genotypes: Suv39h1-deficient (h1KO), Suv39h2-deficient (h2KO), and DKO. Densitometry data were expressed relative to Lamin B1 (LB1) in that lane and then normalized to WT levels. Mean and SEM together with individual data points are shown. Data were statistically analyzed by 1-way ANOVA with Holm-Sidak multiple comparisons test comparing all genotypes with all others. (B) Western blot analysis of H3K9me3 levels from CD45.2⁺ DP thymocytes of Suv39h1-deficient and DKO genotypes (400 000 cells loaded per lane). Mean and SEM together with individual data points are shown. Data were statistically analyzed by Student t test. (C) Representative immunofluorescence staining of heterochromatin protein 1-α (HP1α) and colocalization with DAPI bright spots. Scale bar depicts 2 μm. (D) Quantification of colocalization index of thresholded HP1α staining with DAPI bright spots. Data are shown as individual data points together with mean and SEM. Statistical analysis by Kruskal-Wallis H test with Dunn post hoc test. n = 40, 32, 35, 31, 30, respectively, pooled from at least 3 mice of each genotype. (E) Representative immunofluorescence staining of Fibrillarin, Lamin B1, and DAPI from CD45.2⁺ mouse DP thymocytes and human CD8⁺ T lymphocytes from cord blood and aged (69 and 74 year old) volunteers (representative of 2 donors of each age). Quantification of fibrillarin spot volume (F-G) shows individual nuclei pooled from all samples together with geometric mean and 95% confidence interval. Data in panel F statistically analyzed by Mann-Whitney U test. Data in panel G statistically analyzed by Kruskal-Wallis H test with Dunn post hoc test. All microscopy images were captured on a Zeiss 880 LSM Laser Scanning Microscope with Airyscan using a 63× Oil Immersion Objective with Numerical Aperture of 1.4.