Research Paper Advance Articles

Figure 2. PTGS2 induces and maintains NHEK senescence. (A) NHEKs (donor 4F0315) were treated with NS398 at 5 or 10 µM or DMSO every 48 h. Left panel: During the treatment, cells were passaged when reaching 70% confluence, counted, and the number of population doublings was calculated (see Methods). The experiment was performed in triplicate, each point representing the mean of three counts. Significant differences between DMSO control and 5 or 10 µM NS398 treatment are indicated. Right panel: Senescent NHEKs were treated as in (A), then, a SA-β-Gal assay was performed seven days post-treatments. (B) Senescent NHEKs (donor K23FC1) were transfected with a pool of 4 siRNAs targeting PTGS2, or with non-target siRNAs. Left panel: Evaluation of the efficacy of the siRNAs by Western Blot. GAPDH was used as a loading control. Right panel: Four days after transfection, a SA-β-Gal assay was performed. The bars represent the mean ±SD of three counts of blue cells (^**p < 0.01). (C) Senescent NHEKs (donor K23FC1) were treated with NS398 or rofecoxib at the indicated concentrations for four days. Then, a SA-β-Gal assay was performed. The bars represent the mean ±SD of three counts of blue cells (^*p < 0.05; ^**p < 0.01). (D) ELISA assays for measuring the amounts of GM-CSF and G-CSF in the conditioned media (secreted) of exponentially growing, pre-senescent and senescent NHEKs (donor K40FH1) treated or not with NS398 (5 µM) for 16 hrs. Measures were performed in triplicate. The bars represent the mean ± SD of three counts. Significant differences are indicated with asterisks with ^*p < 0.05; ^**p < 0.01; ^***p < 0.001.