Fig 1: Application of the AgeScore on Progeria syndromes. (A) Cell Model for validation of proposed AgeScore in fibroblasts of donors with Progeria Syndrome in comparison to young/mid-age donors. (B) Amount of ?H2A.X foci per cell examined by IF stainings to investigate DSBs. There were an increase in ?H2A.X foci in all cells of the premature aging syndromes. [n = 3–4 (each n tested >15 cells), mean ± SEM,*p < 0,05, **p < 0.001, ***p < 0,0001, one-way ANOVA] (C) CTCF of H3K9Me3 in human fibroblasts of Progeria patients. There was a decrease in H3K9Me3 expression in donor fibroblasts of HGPS patients (HGPS 1 and HGPS 2). [n = 3–4 (each n tested >15 cells), mean ± SEM, *p < 0,05, **p < 0.001, ***p < 0.0001, one-way ANOVA] (D) Telomere length were measured by MM-qPCR in comparison to a standard of healthy mixed aged individuals. There were no change in telomere length of human fibroblasts of progeria syndrome patients in comparison to age-matched controls. [n = 3, mean ± SEM, *p < 0.05, **p < 0.001, ***p < 0.0001, one-way ANOVA] (E) RNA expression levels measured by qRT-PCR of p21 and p16. Activation of cell cycle arrest were detected in WS by upregulated RNA expression levels of p16. [n = 3-4, mean ± SEM *p < 0.05, **p < 0.001, ***p < 0.0001, one-way ANOVA] (F) Quantification of SA-ßGAL positive cells by X-Gal staining. Donor fibroblasts from premature aging syndromes showed a significant rise of SA- ßGAL positive cells. [n = 3–4 (each n tested >15 cells), mean ± SEM, *p < 0.05, **p < 0.001, ***p < 0.0001, one-way ANOVA] (G) CTCF of Lamin B1 expression. A significant decrease in Lamin B1 expression was observed in HGPS1, HGPS 2 and WS1. [n = 4 (each n tested >15 cells), mean ± SEM, *p < 0,05, **p < 0,001, ***p < 0.0001, one-way ANOVA] (H,I) Determination of cytokine secretion (IL-6, IL-8) in conditioned medium of human fibroblasts by ELISA. From studied progeria donor fibroblasts, HGPS1 and HGPS 2 displayed an increase in IL-8 concentration in comparison to the mean of age-matched controls. [n = 3-4, mean ± SEM, *p < 0.05, **p < 0.001, ***p < 0.0001, one-way ANOVA] (J) Change in nucleus morphology shown as nucleus area (pixel units). Nucleus area was significantly increased in all cells of progeria syndrome patients in comparison to age-matched controls. [n = 6–8 (each n tested >15 cells), mean ± SEM *p < 0.05, **p < 0.001, ***p < 0.0001, one-way ANOVA] (K) Calculated AgeScore of tested human fibroblasts. The AgeScores of progeria syndrome patients (HGPS 1 and 2, WS 1 and 2) are higher in comparison to the age-matched controls.
Fig 2: Examination of the AgeScore in human fibroblasts under replicative senescence. (A) Cell Model for validation of proposed AgeScore, fibroblasts from young, mid-age and old donors were replicative aged. (B–I) X¯ = Mean value of all young donors. (B) Quantification of ?H2A.X in fibroblasts in high passages (p > 35) in comparison to the mean of all Younglow. Amount of ?H2A.X foci per cell increased in all cells in high passages. [n = 4 (each n tested >15 cells), mean ± SEM,*p < 0.05, **p < 0.001, ***p < 0.0001, one-way ANOVA] (C) Quantification of H3K9Me3 by IF staining. CTCF displayed a significant decrease of H3K9Me3 expression in high passages of Young 4, Midage 1, Midage 3 and Old 3 compared to grouped Younglow. [n = 4 (each n tested >15 cells), mean ± SEM,*p < 0.05, **p < 0.001, ***p < 0.0001, one-way ANOVA] (D) Telomere length was measured by MM-qPCR in comparison to a standard of healthy mixed aged individuals. In comparison to grouped Younglow, high passages of Young 1, Young 2, Midage 3 and Old 3 displayed a shortening of telomere length. [n = 3, mean ± SEM, *p < 0.05, **p < 0.001, ***p < 0.0001, one-way ANOVA] (F) RNA expression levels by qRT-PCR of p21 and p16. Cell cycle arrest was detected by upregulation of p21 and p16 in Young 1high, Midage 1high, Old 1high and Old 3high [n = 4, mean ± SEM *p < 0,05, **p < 0.001, ***p < 0.0001, one-way ANOVA] (G) Investigation of cytokine secretion (IL-6, IL-8) in conditioned medium by ELISA. SASP activation was elevated in all fibroblasts under replicative senescence in comparison to the mean of all Younglow [n = 3-4, mean ± SEM, *p < 0.05, **p < 0.001, ***p < 0.0001, one-way ANOVA] (E) Quantification of nucleus area by pixel units. Nucleus area was significantly increased in all high passages of human fibroblasts in comparison to grouped low passages of Younglow. [n = 8 (each n tested >15 cells), mean ± SEM *p < 0.05, **p < 0.001, ***p < 0.0001, one-way ANOVA] (H) Quantification of SA-ßGAL positive cells by X-Gal staining. Amount of positive SA-ßGal stained cells was significantly increased in all cells in high passage in comparison to the mean of all Younglow. [n = 4 (each n tested >15 cells), mean ± SEM, *p < 0,05, **p < 0.001, ***p < 0.0001, one-way ANOVA] (I) Quantification of Lamin B1 expression by IF staining. A significant decrease in Lamin B1 expression was observed in low passages of Midage 1, Midage 2 and in all high passages [n = 4 (each n tested >15 cells), mean ± SEM, *p < 0,05, **p < 0.001, ***p < 0.0001, one-way ANOVA] (J) Calculated AgeScore. The AgeScore increases in cells with replicative senescence.
Fig 3: Examination of the AgeScore in premature aged human fibroblasts of young donors. Data for prematurely aged fibroblasts (induced with Doxycyclin) are marked in red. (A) For validation of the proposed AgeScore, young donor fibroblasts were premature aged by dox-inducible Progerin overexpression. (B) Representative IF images of ?H2A.X and GFP-Progerin signal in absence and presence of doxycycline (1 µg/mL) after 4 days. Amount of ?H2A.X foci per cell increased in cell lines with GFP-Progerin expression (Young 4, Young 5) in comparison to non-induced fibroblasts. [n = 3 (each n tested >15 cells), mean ± SEM,*p < 0.05, **p < 0.001, ***p < 0.0001, one-way ANOVA, scale bar = 10 µM] (C) Representative IF images of H3K9Me3 and GFP-Progerin signal in absence and presence of doxycycline (1 µg/mL) after 4 days (merge includes magenta = Vimentin). CTCF displayed no change in prematurely aged young donor of H3K9Me3 [n = 3 (each n tested >15 cells), mean ± SEM,*p < 0.05, **p < 0,001, ***p < 0.0001, one-way ANOVA, scale bar = 10 µM] (D–I) X¯ = Mean value of all young donors. (D) Telomere length was measured by MM-qPCR in comparison to a standard of healthy mixed aged individuals. In comparison to non-induced cells, Young 4 and Young 5 with progerin expression displayed a shorting of telomere length. [n = 3, mean ± SEM, *p < 0,05, **p < 0.001, ***p < 0.0001, one-way ANOVA] (E) RNA expression levels by qRT-PCR of p21 and p16 was measured to investigate activation of cell cycle arrest in human fibroblasts. Young 1, Young 4 and Young 5 with progerin expression displayed a raise of relative RNA expression level of p16 in comparison to non-induced controls. [n = 3, mean ± SEM *p < 0.05, **p < 0.001, ***p < 0.0001, one-way ANOVA] (F) Representative IF images and quantification of SA-ßGAL positive cells by X-Gal staining. All three young donor cell lines with induced GFP-Progerin expression showed an increase in amount of positive stained SA-ßGAL cells. [n = 3 (each n tested >15 cells), mean ± SEM, *p < 0.05, **p < 0.001, ***p < 0.0001, one-way ANOVA] (G) Representative IF images and quantification of nucleus are (pixel units) in prematurely aged human fibroblast. Nucleus area was significantly increased in all three young donor fibroblasts with induced GFP-Progerin. [n = 6 (each n tested >15 cells), mean ± SEM *p < 0.05, **p < 0,001, ***p < 0.0001, one-way ANOVA, scale bar: 10 µM] (H) Determination of cytokine secretion (IL-6, IL-8) in conditioned medium of human fibroblasts with GFP-Progerin expression in comparison to non-induced cells by ELISA. All prematurely aged fibroblasts showed a significant increase in IL-6 and IL-8 concentrations in comparison to grouped controls. [n = 3-4, mean ± SEM, *p < 0,05, **p < 0.001, ***p < 0.0001, one-way ANOVA] (I) Representative IF images and quantification of Lamin B1 IF staining. A significant decrease in Lamin B1 expression was observed in all three prematurely aged human fibroblasts [n = 3 (each n tested >15 cells), mean ± SEM, *p < 0.05, **p < 0.001, ***p < 0,0001, one-way ANOVA] (J) Calculated AgeScore for prematurely aged fibroblasts of young donors in comparison to non-induced controls.
Fig 4: Establishment of the age marker-based panel in human fibroblasts of young, mid-age and old donors. (A) Cell Model. For initial experiments, fibroblasts with different donor age was used. (B) Replicative potential of human fibroblasts. Cells were cultured under stable conditions (37°C, 5% CO2) in DMEM medium (with 15% FBS, 1% Pen-Strep). PDL was observed for max. 150 days. Fibroblasts from young (Young 1–5) and mid-age (Midage 1–3) donors display healthy growth. In contrast, fibroblasts from old donors (Old 3, Old 4, Old 5 and 6) showed slower growth except for Old 1 and Old 2. (C–K) X¯ = Mean value of all young donor fibroblasts. (C) Amount of ?H2A.X foci per cell examined by IF staining to investigate DSBs in human fibroblasts. Cells of very old individuals (Old 4 and 5) displayed an increase in ?H2A.X foci in comparison to the mean of all young donors. Mid-age and old (Old 1, Old 2, Old 3 and Old 6) donor fibroblast showed no DSBs. [n = 3–4 (each n tested >15 cells), mean ± SEM,*p < 0.05, **p < 0,001, ***p < 0.0001, one-way ANOVA] (D) CTCF of H3K9Me3 in human fibroblasts. Donor fibroblasts from old individuals (Old 2, Old 4, Old 5 and Old 6) displayed a decrease in CTCF of H3K9Me3 compared to the mean of young donors. There were no changes in CTCF of H3K9Me3 in mid-age cells (Midage 1–3). [n = 3–4 (each n tested >15 cells), mean ± SEM, *p < 0.05, **p < 0.001, ***p < 0.0001, one-way ANOVA] (E) Telomere length was measured by MM-qPCR in comparison to a standard of healthy mixed aged individuals. In comparison to grouped young donor cells, Old 6 displayed a shortening of telomere length. [n = 3, mean ± SEM, *p < 0.05, **p < 0.001, ***p < 0.0001, one-way ANOVA] (G) RNA expression levels measured by qRT-PCR of p21 and p16 was examined to investigate activation of cell cycle arrest. Old 4 and Old 5 displayed an increase in RNA expression level of p21. All other cells did not show altered RNA expression levels. [n = 3-4, mean ± SEM *p < 0.05, **p < 0.001, ***p < 0.0001, one-way ANOVA] (F) Quantification of SA-ßGAL positive cells by X-Gal staining. In comparison to grouped young donor cells, Midage 3 and all old human fibroblasts (Old 1–6) displayed a significant increase of SA- ßGAL positive cells. [n = 3–4 (each n tested >15 cells), mean ± SEM, *p < 0,05, **p < 0.001, ***p < 0.0001, one-way ANOVA] (H) CTCF of Lamin B1 expression. A significant decrease in Lamin B1 expression could be observed in mid-age (Midage 1 and Midage 3) and old (Old 1–5) cells compared to the mean of young donor fibroblasts. [n = 4 (each n tested >15 cells), mean ± SEM, *p < 0.05, **p < 0.001, ***p < 0.0001, one-way ANOVA] (I, J) Determination of cytokine secretion (IL-6, IL-8) in conditioned medium by ELISA. In comparison to grouped young donor cells, old donor fibroblasts (Old 1, Old2, Old 3, Old 4, and Old 6) showed an increase in levels of IL-6. An increase in concentration with respect to IL-8 could be observed in Midage 1 and Old 4 in comparison to grouped controls. [n = 3-4, mean ± SEM, *p < 0.05, **p < 0,001, ***p < 0.0001, one-way ANOVA] (K) Change in nucleus morphology shown as nucleus area (pixel units) in human fibroblast. Nucleus area is significantly increased in very old donor fibroblasts (Old 4 and 5) compared with grouped young controls. [n = 6–8 (each n tested >15 cells), mean ± SEM *p < 0.05, **p < 0.001, ***p < 0.0001, one-way ANOVA].
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