Figure 1: Premature cellular senescence in Down syndrome primary fibroblasts.

All experiments were conducted in primary fibroblasts from age- and sex-matched donors either without Down syndrome (NDS, GM00696 and GM05659) or with Down syndrome (DS, AG06872 and AG05397) as described in Experimental. Graphs are labeled DS or NDS. A. Down syndrome fibroblasts exhibit a premature senescence phenotype. Senescence-associated β-galactosidase (SA-β-Gal) activity was measured in primary fibroblasts from donors with or without Down syndrome as described in Experimental. A minimum of 300 cells was counted in random fields by a technician blinded to genotype to quantify the proportion of SA-β-Gal-positive cells. Quantification is presented as the average of three independent experiments ± SEM and is expressed as a percent of positive cells [(SA-β-Gal-positive cells/total cells)*100]. Data is expressed as mean ± SEM for three independent pooled experiments. *Value significantly different from control at p < 0.01. B. Upregulation in p16 in Down syndrome primary fibroblasts. p16 transcript abundance was determined by quantitative RT-PCR and normalized to GAPDH as described in methods. Data is expressed as mean ± SEM for three independent experiments. *Value significantly different from control at p < 0.01. C. HMGB1 is excluded from the nuclear fraction in Down syndrome primary fibroblasts. Nuclear and cytoplasmic fractions were extracted from cells, separated on 10% TGX Stain-free precast gels (BioRad) and transferred to PVDF. Membrane was incubated in 1:500 dilution of HMGB1 (Sigma, Cat# H9664). HMGB1 in nuclear and cytoplasmic fractions was normalized to total protein as described in Experimental. Normalization (loading control) was done for both nuclear and cytoplasmic fractions by calculating optical density of HMGB1/total protein. Graph is labeled for genotype (DS or NDS) and fraction (NF, nuclear fraction; CF, cytoplasmic fraction). Data is expressed as mean ± SEM for three independent experiments from pooled averages. *Value significantly different from control at p < 0.01. D. Primary Down syndrome fibroblasts exhibit reduced DNA polymeraseβ transcript abundance. Gene expression was evaluated in primary fibroblasts from donors with or without Down syndrome as described in Experimental. POLβ transcript levels were determined by quantitative real-time RT-PCR and normalized to GAPDH. Data are presented as the average of pooled samples from each genotype ± SEM. *Values significantly different from control (NDS) at p < 0.01.