PMC

Skin biopsies (2mm) were obtained from young (21-30 years) and elderly (>80 years) sun-protected buttock skin. PGE₂ levels were quantified by substrate enzyme immunoassay and normalized to DNA content of corresponding skin samples. (N=10, *p<0.05).

(a) Relative PTGES1 mRNA expression in human buttock skin was determined by cDNA microarray analysis. Results show positive correlation of PTGES1 mRNA level and age of donors (N=62, p=2.6 × 10⁻⁷, r=0.60). (b) PTGES1 mRNA expression in buttock skin was determined by qPCR. (N=40, p=1.38 10⁻⁷, r=0.73). The subjects used in microarray analysis were different from the subjects used in qPCR analysis.

Immunohistochemistry of PTGES1 protein in young (21-30 years old) and aged (>80 years old) sun-protected buttocks human skin. Left panels: representative immunostaining in upper and lower dermis. PTGES1 protein staining is brownish, and nucleus counterstaining with hematoxylin is blue. Arrows point to PTGES1 positively stained cells. Right panel: Percentage of total dermal stromal cells that were PTGES1 positive. (N=6, *p<0.01) Scale=50µm

(a) Fibroblasts were cultured with the addition of PGE₂ (10nM) or vehicle (DMSO) for 24 hours. Type I procollagen protein levels in cell lysates were determined by Western blot, normalized to β-actin. Inset shows representative Western blots. (N=5, *p<0.05). (b) Skin samples were incubated in serum-free α-MEM media with the addition of diclofenac (10 µM) or vehicle (DMSO) for 16 hours. Type I collagen mRNA levels were quantified by qPCR and normalized to 36B4 mRNA levels (N=6). Type I procollagen protein levels in conditioned media were quantified by EIA. (N=3, *p<0.05).

(a-d) Fibroblasts obtained from young (21-30 years) individuals seeded on micropost arrays. (a) Scanning electron microscopy of fibroblasts. Micrographs are representative of three experiments examining >60 fibroblasts. (b) Quantification of traction force (nanonewton, nN) for 10 fibroblasts/condition (N=3, *p<0.001). (c) COX2 and PTGES1 mRNA levels was determined by qPCR (N=3, *p<0.05). (d) PGE₂ levels in conditioned-medium were quantified by EIA, and normalized to cell number (N=3, *p<0.05). (e-f) Fibroblasts cultured from young skin were seeded on type I collagen-coated hydrogels with low or high compliance. (e) PTGES1 and COX2 mRNA levels were quantified by qPCR. (N=3, *p<0.05). (f) PGE₂ levels in conditioned media quantified by EIA. (N=3, *p<0.05).

Dermis and epidermis of frozen skin sections were separated and collected using laser capture microscopy. Relative (a) PTGES1 and (b) COX2 mRNA levels in the epidermis and dermis of young and aged individuals were determined by qPCR normalized to 36B4 mRNA. N=6, *p<0.05. (c-d) Cells were released from fresh sun-protected skin samples by collagenase digestion. Released cells were fractionated by immuno-affinity magnetic beads to separate fibroblasts for other cell types. Total RNA was extracted from isolated cells. (c) PTGES1 mRNA levels were determined in fibroblast-enriched and fibroblast-depleted cells (N=4, *p<0.05). (d) PTGES1 mRNA levels were determined in fibroblast-enriched cells isolated from skin samples from young and aged individuals. (N=5-7, *p<0.05).