Research Paper Volume 16, Issue 21 pp 13201—13224

Figure 4. EP4 receptors mediate senescence of epidermal keratinocytes. (A) The mRNA levels of EPs were detected by RT-PCR in extracts from exponentially growing and senescent NHEKs (donor 4F0315) and in NHDFs at the replicative senescence plateau. Actin levels were used as control. The number of population doublings for NHDFs and NHEKs are indicated. (B) Immunofluorescence detection of EP1 and EP4 (green) performed in sections of skin samples from human young and old healthy subjects (see Material and Method). Cell nuclei were detected by DAPI staining (blue). Representative confocal microscopy images of the epidermis of a young (37 years old) and an elderly donor (83 years old). Positive staining of EP1 and EP4 (white arrows) are shown. Bars represent 20 µm. (C) Scheme of PGE₂ receptors and their agonists/antagonists used in the following experiments. (D) NHEKs (donor K40FH1) at the exponential growth phase were treated with the EP1 or EP4 agonists (Iloprost, L-902,688 and Rivenprost, respectively at 100 ng/mL, 1 µM and 100 nM) for 4 days. The percentage of SA-β-Gal-positive cells was determined 4 days after the beginning of treatment. SA-β-Gal-positive cells were counted in at least 3 different microscopic fields. The bars represent the mean ± SD of at least 3 counts (^**p < 0.01). (E) Senescent NHEKs (donor K23FC1) were treated with EP1 and EP4 antagonists (AH6809 and L-161,982, respectively at 10 µM and 1 µM). The percentage of SA-β-Gal-positive cells was determined 4 days after the beginning of treatment. SA-β-Gal-positive cells were counted in at least 3 different microscopic fields. The bars represent the mean ± SD of at least 3 counts (^*p < 0.05). (F) NHEKs (donor K40FH1) at the exponential growth phase were treated as in (D), the amounts of GM-CSF in the conditioned media (secreted) were measured by an ELISA assay. Measures were performed in triplicate. The bars represent the mean ± SD. Significant differences are indicated with asterisks with ^*p < 0.05 and ^***p < 0.001. (G) NHEKs (donor K40FH1) at the exponential growth phase were treated as in (E), and the amount of GM-CSF in the conditioned media (secreted) was measured by an ELISA assay. Measures were performed in triplicate. The bars represent the mean ± SD. Significant differences are indicated with asterisks with ^*p < 0.05.