Research Paper Volume 16, Issue 21 pp 13201—13224

Figure 5. Loss of PTGS2 activity reduces preneoplastic senescence escape of NHEKs. (A) Left panel: Scheme depicting the experimental process. Right panel: representative phase contrast microscopy image of what was counted as a clone in the following experiments. (B) Senescent NHEKs (donor K67JA1) were subjected to PTGS2 siRNAs vs. non-target control siRNAs. After four- or six-days post-transfection, emerging clones were manually counted under microscopic examination after fixation and coloration with crystal violet. (C) Senescent NHEKs (donor K23C1) were subjected to PTGS2 pharmacological inhibition by NS398 or Rofecoxib at the indicated concentrations. Four- or six-days post-treatment, emerging clones were manually counted as in B. (D) Senescent NHEKs (donor KNBMC1) were treated or not with 10 or 100 nM PGE₂ 4-times a day. After four days of treatment, the number of emerging clones was counted as in B. (E) Senescent NHEKs (donor K40FH1) were treated with the EP1 or EP4 agonists (Iloprost and Rivenprost at the indicated concentrations). After four days of treatment, the number of emerging clones was counted as in B. (F) Senescent NHEKs (donor K40FH1) were treated with EP1 and EP4 antagonists (AH6809 and L-161,982, respectively at 10 µM and 1 µM). After four days of treatment, the number of emerging clones was counted as in B. In (B–F), the bar chart represents the mean ± SD of the 3 independent measures (^*p < 0.05; ^**p < 0.01). In (B–F), each result is representative from 3 independent experiments, with each experiment corresponding of three or four technical replicates.