Research Paper Volume 16, Issue 21 pp 13201—13224

Figure 1. PTGS2 is up-regulated during NHEK in vitro senescence and skin aging. (A) The mRNA levels of PTGS2 were measured by RT-qPCR in extracts from exponentially growing, pre-senescent and senescent NHEKs (donor 4F0315). PTGS2 levels were normalized to EAR levels. The bars represent the mean ±SD (p < 0.05; ^**p < 0.01). (B) PTGS2, MnSOD, PCNA, and GPX4 protein levels were evaluated by Western Blot in extracts from exponentially growing, pre-senescent and senescent NHEKs (upper panel: donor 4F0315; lower panel: donor K40FH1). The gel was equicharged with extracts from an equal number of cells. The equicharge was verified a posteriori by detecting the levels of actin and GAPDH. (C) Exponentially growing and senescent NHEKs (donor K3MC1) were fixed and processed for immunofluorescence detection of endogenous proteins PTGS2 (red) and XRCC1 (green). Cell nuclei were detected by DAPI staining (blue). Upper panel: Representative confocal photomicrographs of PTGS2 inmmunostaining and XRCC1 foci (white arrows). Bars represent 20 µm. Lower panel: XRCC1 foci were quantified. Measures were done in five independent microscopic fields for a total of at least 100 cells for each condition. The histogram represents the average ± S.D. of five counts. Results are representative of at least two independent experiments. (D) Immunofluorescence detection of PTGS2 (red) performed in sections of skin samples from human young (n = 4) and old (n = 5) healthy subjects (see Material and Method) (^*p < 0.05). Cell nuclei were detected by DAPI staining (blue). Upper panel: representative confocal microscopy images for epidermis and dermis of a young (37 years old) and an elderly donor (85 years old). The squares delimit the below images at higher magnification. Bars represent 40 µm. Lower panel: scatter dot plots indicating the mean fluorescence intensity in cells of the basal layer. A minimum of 25 cells per sample were selected individually in order to obtain a mean fluorescence for each donor. The horizontal black lines denote median values and the boxes the interquartile ranges (IQR). Vertical lines extend from max value (upper quartile + 1.5*IQR) to min value (lower quartile – 1.5*IQR).