Research Paper Advance Articles

Figure 3. PGE₂ contributes to the establishment and maintenance of senescence in NHEKs. (A) PTGS2, PTGES1, PTDGS, and PGFS mRNA levels were measured by RT-qPCR in NHEKs (donor K67FA1) at the exponential growth phase or at the senescence plateau and were normalized to EAR levels. Results are presented in log 10 scale relative to PTGS2 expression in exponentially growing NHEKs (^*p < 0.05; ^***p < 0.001). (B) The amount of PGE₂ and PGF₂ in the culture media (secreted) of exponentially growing or senescent NHEKs (donor K67FA1) were measured by a competitive assay (see Material and Methods). Measures were performed in triplicate. The bars represent means ± SD. (C) Senescent NHEKs (donor K40FH1) were transfected with a pool of 4 siRNAs targeting PTGES1 or PGFS, or with non-target siRNAs (siCtrl). Four days after transfection, a SA-β-Gal assay was performed. The bars represent the mean ± SD of three counts (^*p < 0.05; ^***p < 0.001). (D) The amounts of IL-10, GM-CSF and G-CSF in the conditioned media (secreted) were measured by ELISA assays in NHEKs (donor K40FH1) treated as in (C). Measures were performed in triplicate. The bars represent the mean ± SD. Significant differences are indicated with asterisks with ^*p < 0.05. (E) Pre-senescent NHEKs (donor K23FC1) were treated or not with 10 nM to 10 µM PGE₂ 4-times a day. The percentage of SA-β-Gal positive cells 4 day after the beginning of the treatment was determined. The bars represent the mean ± SD of three counts (^*p < 0.05; ^**p < 0.01). (F) Pre-senescent NHEKs (donor K23FC1) were treated as in (E), the number of cells was then determined. The bars represent the mean ± SD of three counts (^**p < 0.01).