180 JOURNAL OF COSMETIC SCIENCE
However, in our hands the treatment with PLE induced these signals without a detectable
increase of actual DNA damage. This indicates that PLE is capable of boosting the G2
DNA repair machinery in a direct manner by mechanisms still to be elucidated. The p53 is
referred to as the guardian of the genome due to its capacity to correctly coordinate the cell
cycle phases. Altogether, our results indicate that PLE reinforces the orchestral execution of
the cell cycle. This is consistent with the lower index of expression of keratin K16, a marker
of epidermal hyperproliferation and stress.34
Figure 4. PLE reduces the proportion of keratinocytes undergoing stress-induced terminal differentiation.
(A) Left, representative flow cytometry profiles of the levels of involucrin (solid red line) in primary human
keratinocytes treated or not with PLE, as indicated, for 8 days. Dotted black line is negative antibody control.
Right, quantification of the percent of involucrin-positive cells according to the gate shown on the left.
Data are mean ± SD of duplicate samples representative of two independent experiments. **p 0.01 (B)
representative immunofluorescence images of untreated or PLE-treated keratinocytes, as indicated, for 5 days.
Red is involucrin (arrows) and blue is nuclear DNA stained with 4′,6-diamidino-2-phenylindole. Broken line
outlines the border of the colonies. Scale bar: 50 µm and (C) representative flow cytometry profiles of keratin
K16 expression (red line) in human keratinocytes treated or not with PLE for 8 days. Dotted line as in A. Bar
histogram shows the percent of K16-positive cells according to the gate in the left plots. Data are mean ± SD
of duplicate samples. **p 0.01. FLH1: Fluorescence channel 1 Height.

181 POLYPODIUM LEUCOTOMOS EXTRACT
Figure 5. Pretreatment with PLE boosts the γH2AX signal and attenuates the differentiation response
induced by sublethal UV irradiation. (A) Top, flow cytometric analyses of DNA content determined by
propidium iodide (PI) of epidermal keratinocytes, treated or not, as indicated, with PLE for 6 days and
irradiated with sublethal doses (25 mJ/cm2) of UVB light. Cells were analyzed 48 hours postirradiation.
Bottom, bar histogram shows the quantification of the percentage of cells in G1 and S +G2/M normalized
to untreated, nonirradiated cells (B) top, flow cytometry profiles of the levels of γH2AX (solid magenta line)
in epidermal keratinocytes treated as in A. Secondary antibody staining control is shown as a dotted black
line. Bottom, quantification of percent of γH2AX-positive cells according to the gate shown on the plots
above. Data are mean ± SD of duplicate samples representative of two to three independent experiments.
*p 0.05, **p 0.01 (C) flow cytometry profiles of the levels of involucrin (solid red line) in epidermal
keratinocytes as in A. Secondary antibody staining control is shown as a dotted black line. Bottom: bar
histogram: quantification of the percent of involucrin-positive cells according to the gate on the plots above.
Data are mean ± SD of duplicate samples representative of two independent experiments. **p 0.01 and
(D) photographs of phase contrast microscopy of cells in A–C, as indicated. Note the differentiated phenotype
in the UV-treated culture, with large cells and shedding, compared with the less differentiated culture when
pretreated with PLE. Scale bar: 50 µm.