Fig 1.
Experimental design, growth curves and transition into senescence.
(A) Experimental plan of culturing fibroblast cell strains derived from a single vial and maintained in culture as triplicates from an early PD until senescence at late PDs. (B) Growth curve of five different fibroblast cell strains (MRC-5, BJ, WI-38, IMR-90, HFF) derived from a single vial and maintained in culture as triplicates from an early PD until senescence at late PDs. Data points of all measurements are displayed (not the mean). (C) Percentage of SA-β Gal positive cells at different time points of their growth in culture in the five fibroblast strains derived from a single vial. Each curve is measured in triplicate, the mean value is displayed with error bar (± S.E).
Fig 2.
Fifty most up-regulated genes with age in each of the five fibroblast strains.
The red background represents genes commonly up-regulated with age in foreskin fibroblasts (HFF, BJ) and green background reveals genes commonly up-regulated among all the three embryonic lung fibroblasts (MRC-5, IMR-90, WI-38) as well as between the fibroblasts derived from female donors (IMR-90 and WI-38) among the hundred most differentially regulated genes with age.
Fig 3.
Fifty most down-regulated genes with age in each of the five fibroblast strains.
The red background represents genes commonly down-regulated with age in foreskin fibroblasts (HFF, BJ) and green background reveals genes commonly down-regulated among all the three embryonic lung fibroblasts (MRC-5, IMR-90, WI-38) as well as between the fibroblasts derived from female donors (IMR-90 and WI-38) among the hundred most differentially regulated genes with age.
Fig 4.
Significantly differentially regulated pathways with age across the five fibroblast strains.
The most significantly up- and down-regulated pathways across the five fibroblast strains retrieved by performing gene set enrichment analysis by applying the R package gage (Generally Applicable Gene-set Enrichment) in combination with all annotated KEGG pathways separately for each of the five fibroblast strains.
Fig 5.
The differentially regulated pathways across the five fibroblast strains and the public data sets.
The most significantly up- and down-regulated pathways across the five fibroblast strains and the nine selected public data sets retrieved by performing gene set enrichment analysis. Pathways highlighted with blue rectangles have similar regulation pattern across all the 14 datasets.
Fig 6.
Recombinant human SFRP4 protein treatment in human foreskin fibroblasts (HFF).
(A) Percentage of SA-β Gal positive cells at different time points up until 10 days in HFF strains (PD = 18) maintained in conditioned medium containing different concentrations of recombinant human SFRP4 (rSFRP4) proteins (0, 5, 10, 15 μg/ml). Samples specified control were untreated fibroblasts and those specified treated with 0 μg were treated with PBS containing 0.1% Bovine serum albumin instead of rSFRP4. The bars indicate the mean ± S.D. Values statistically different from their controls (Student’s t-test, 95% confidence level) are indicated with an asterix. ** p < 0.01, *** p <0.001- Significantly different compared to controls. n = 3 (B) Results of quantitative immunoblots showing protein expression levels of p16 and p21 in HFF strains treated with 15 μg/ml recombinant human SFRP4 proteins compared to controls and HFF strains treated with 0 μg SFRP4. n = 3 (C) Immunoblot shows protein expression levels of SFRP4 in control (CON; PD = 18), senescent (SEN; PD = 74) and young fibroblasts (PD = 18) maintained in culture supplemented with conditioned medium containing 0, 10 or 15 (μg/ml) rSFRP4 proteins for 10 days. n = 3 (D) Immunoblot shows protein expression levels of β-Catenin in control (CON; PD = 18), senescent (SEN; PD = 74) and young fibroblasts (PD = 18) maintained in culture supplemented with conditioned medium containing 0, 10 or 15 (μg/ml) rSFRP4 proteins for 10 days. n = 2.
Fig 7.
Genes belonging to the cytokine-cytokine receptor interaction pathway which are significantly up- (green) or down- (red) regulated (log 2 fold change > 1) across the five fibroblast strains during replicative senescence.
Genes highlighted with boxes colored red+green are up-regulated in certain and down-regulated in the other fibroblast strains.