Figure 2. Chemerin is an upregulated SASP factor in human dermal fibroblasts.
(A) Graph demonstrating the relative RARRES2 (Chemerin gene) mRNA expression in senescent (SEN) vs. young (YNG) fibroblast of different strains (FF95, FFRa and FFPia) as defined by qRT-PCR. Data are normalized to the expression level of RARRES2 in keratinocytes, confirming that the senescent fibroblasts display the highest, and the cSCC cell lines (SCL-1, SCC12-B2, SCC-13) display the lowest RARRES2 transcripts, respectively. Date are shown as mean ± S.D for one of three independent experiments of biological replicates (n = 3); *p < 0.05, **p < 0.01 and ***p < 0.001 calculated by Bonferroni post hoc test after ANOVA. (B) Chemerin secretion was analyzed in the above mentioned cells (normalized to 5 × 106 cells/ml) using ELISA. Data are shown as mean ± S.E.M for three independent experiments; *p < 0.05, **p < 0.01 and ***p < 0.001 calculated by Bonferroni post hoc test after ANOVA. (Note that due to low standard deviations of some measurements, error bars are not visible for all data points.) (C) Representative photomicrographs of paraffin-embedded human skin sections co-immunostained with anti-FSP-1 antibody in green and anti-Chemerin antibody in red, depicting higher abundance of Chemerin in skin dermal fibroblasts of aged (70-year old), compared to young (23-year old) donors. Nuclei were DAPI-counterstained (blue). Appropriate isotype controls were used to determine the background. Scale bars = 50 μm at ×400 magnification; Dashed lines delineate epidermis (E) from dermis (D). Orange arrows point to the Chemerin-positive fibroblasts. Orange boxes depict the magnified area. (D) Graph representing the quantification of Chemerin-positive fibroblasts (shown by FSP-1 marker) in the skin dermis of old healthy individuals (76 ± 10 year, n = 15 donors) and young (21 ± 8 year, n = 13 donors) calculated from minimum 5 technical replicates. ***p < 0.001 by two-tailed student t-test.