Figure
2.
PLE
delays
progression
of
epidermal
keratinocytes
through
the
G2-repair
phase
of
the
cell
cycle.
(A)
Representative
flow
cytometry
analyses
of
DNA
content
determined
by
propidium
iodide
(PI)
staining
of
cultured
primary
keratinocytes,
treated
or
not
with
PLE
for
8
days
as
indicated.
Quantitation
of
the
percent
of
cells
in
the
G1
or
the
G2/M
phase
of
the
cell
cycle
is
shown
on
the
right
histograms
(B)
representative
flow
cytometry
analyses
of
light
scatter
parameters
of
cultured
primary
keratinocytes,
treated
or
not
with
PLE
for
8
days,
as
indicated.
The
black
circle
corresponds
to
proliferative
cells
displaying
a
larger
size
typical
of
cells
in
the
DNA
repair
(R)
G2/M
phase
of
the
cell
cycle
(cells
are
twice
as
big).
Quantitation
of
cells
in
this
region
is
shown
on
the
right.
Data
represent
mean
±
SD
of
duplicate
samples,
representative
of
three
independent
experiments.
**p
0.01.
SSC:
Side
Scatter
FSC:
Forward
Scatter.
177 POLYPODIUM LEUCOTOMOS EXTRACT

178 JOURNAL OF COSMETIC SCIENCE
Figure 3. PLE induces DNA repair signals γH2AX and p53 in epidermal keratinocytes independently of
DNA damage. (A) Representative flow cytometry profiles showing levels of γH2AX (solid magenta line) in
primary human keratinocytes, untreated or treated with PLE for 8 days. As a positive control of γH2AX
induction, a separate sample of cells was treated for 24 hours with 1 µM doxorubicin (dotted red line). Dotted
black line is negative isotype antibody control (B) quantitation of the percent of γH2AX-positive cells
according to the gate shown in A (C) Western blot for p53 expression in cells untreated or treated with PLE
for 8 days. Cells treated with Doxorubicin as in A were included as a control for the induction of p53. GAPDH
is used as protein-loading control. Bottom histogram represents the optical density normalized to GAPDH
and relative to control (CT) (D) immunofluorescence for expression of p53 (arrows) of cells treated for 5 days
as indicated. Scale bar: 50 µm. (E) representative nuclear image of comet assay analyses of human epidermal
keratinocytes untreated or treated with PLE for 6 days, as in A and B, to test for chromosomal fragmentation
and (F) quantitation of DNA fragmentation measured by tail length, relative to untreated (UN) control cells
(n =299/302). Scale bar: 10 µm. Data are mean ± SD of duplicate samples representative of two independent
experiments. *p 0.05 ns: not significant (p 0.05).