184 JOURNAL OF COSMETIC SCIENCE
For protein analysis, the following primary antibodies were used: mouse antihistone
H2AX-ser 139 (JBW301, 05636i, MilliporeSigma, Burlington, MA) mouse antikeratin
16 (LL025, sc-53255, Santa Cruz Biotechnology, Dallas, TX) mouse anti-involucrin
(sy3, lab-made, Hudson, 1992). All antibody stainings were controlled using similar
concentration of isotype-negative antibody (mouse IgG, Sigma-Aldrich). Alexa Fluor®
488–conjugated goat anti-mouse IgG antibody was used as secondary antibody (115-
547-003, Jackson ImmunoResearch, West Grove, PA). After staining, samples were
resuspended and filtered through a 70-μm pore nylon mesh (NY-0130/NYLON,
Labopolis, Madrid, Spain) to prevent the presence of aggregates. Analyses were carried
out on a CytoFLEX cytometer (Beckman Coulter, Brea, CA). Ten thousand cells were
gated and acquired.
IMMUNOFLUORESCENCE AND WESTERN BLOTTING
For immunofluorescence, primary human keratinocytes were cultured in 10-mm round
coverslips. Cells were fixed at RT with cold MeOH for 10 minutes or fixed with 3.7%
paraformaldehyde in PBS for 10 minutes at RT and then permeabilized with cold MeOH.
Staining was performed as previously described.16 The following primary antibodies were
used: mouse anti-involucrin (sy3, lab-made, Hudson, 1992) and rabbit anti-p53 (FL393,
SC-6243, Santa Cruz Biotechnology). Alexa Fluor® 488–conjugated goat anti-mouse
or anti-rabbit IgG antibody were used as secondary antibodies (115-547-003, Jackson
ImmunoResearch). DNA was stained with 0,2 µg/ml 4′,6-diamidino-2-phenylindole.
Coverslips were mounted with ProLong Gold Antifade Reagent (P10144, ThermoFisher
Scientific). Images were acquired with a fluorescent microscope (Zeiss, Oberkochen,
Germany) and processed using Axiovison or Zen Blue software.
For Western blotting, cellular pellets were lysed and subjected to SDS-PAGE electrophoresis
and blotting as previously described (soluble protein fraction).16 Rabbit anti-p53 (FL393,
SC-6243, Santa Cruz Biotechnology) was used for p53 detection. An equal amount of
protein was loaded onto the gel, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
was used as loading control. Secondary HRP-conjugated goat anti-rabbit or anti-mouse
IgG antibodies (170-6516, 170-6515, Bio-Rad, Hercules, CA) were used. Membranes were
incubated with enhanced chemiluminescence substrate (Amersham ECL Prime Western
Blotting Detection Reagent, RPN2232, Cytiva, Marlborough, MA) following supplier’s
instructions and imaged with FUSION Solo S-Western Blot &Chemi Imaging system
(Vilber Lourmat, Eberhardzell, Germany).
COMET ASSAYS
Single-cell electrophoresis, or comet assays, were performed as previously described.28
Briefly, cells were gently harvested in the dark and embedded in low-melting-point
agarose. Then a thin layer of the cell suspension was added to slides previously coated
with normal-melting-point agarose. After solidification, slides were incubated for 1 hour
in cold lysis solution (10% DMSO, 1% Triton X-100) and 89% lysis buffer (NaCl 2.5 M,
EDTA 100 mM, Tris 10 mM, NaOH 200 mM, pH 10). Then electrophoresis was carried
out and the samples were fixed with absolute ethanol and stained with RedSafe 1:500
(21141, Intron Biotechnology, Seongnam, South Korea). Images were acquired with a Zeiss
For protein analysis, the following primary antibodies were used: mouse antihistone
H2AX-ser 139 (JBW301, 05636i, MilliporeSigma, Burlington, MA) mouse antikeratin
16 (LL025, sc-53255, Santa Cruz Biotechnology, Dallas, TX) mouse anti-involucrin
(sy3, lab-made, Hudson, 1992). All antibody stainings were controlled using similar
concentration of isotype-negative antibody (mouse IgG, Sigma-Aldrich). Alexa Fluor®
488–conjugated goat anti-mouse IgG antibody was used as secondary antibody (115-
547-003, Jackson ImmunoResearch, West Grove, PA). After staining, samples were
resuspended and filtered through a 70-μm pore nylon mesh (NY-0130/NYLON,
Labopolis, Madrid, Spain) to prevent the presence of aggregates. Analyses were carried
out on a CytoFLEX cytometer (Beckman Coulter, Brea, CA). Ten thousand cells were
gated and acquired.
IMMUNOFLUORESCENCE AND WESTERN BLOTTING
For immunofluorescence, primary human keratinocytes were cultured in 10-mm round
coverslips. Cells were fixed at RT with cold MeOH for 10 minutes or fixed with 3.7%
paraformaldehyde in PBS for 10 minutes at RT and then permeabilized with cold MeOH.
Staining was performed as previously described.16 The following primary antibodies were
used: mouse anti-involucrin (sy3, lab-made, Hudson, 1992) and rabbit anti-p53 (FL393,
SC-6243, Santa Cruz Biotechnology). Alexa Fluor® 488–conjugated goat anti-mouse
or anti-rabbit IgG antibody were used as secondary antibodies (115-547-003, Jackson
ImmunoResearch). DNA was stained with 0,2 µg/ml 4′,6-diamidino-2-phenylindole.
Coverslips were mounted with ProLong Gold Antifade Reagent (P10144, ThermoFisher
Scientific). Images were acquired with a fluorescent microscope (Zeiss, Oberkochen,
Germany) and processed using Axiovison or Zen Blue software.
For Western blotting, cellular pellets were lysed and subjected to SDS-PAGE electrophoresis
and blotting as previously described (soluble protein fraction).16 Rabbit anti-p53 (FL393,
SC-6243, Santa Cruz Biotechnology) was used for p53 detection. An equal amount of
protein was loaded onto the gel, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
was used as loading control. Secondary HRP-conjugated goat anti-rabbit or anti-mouse
IgG antibodies (170-6516, 170-6515, Bio-Rad, Hercules, CA) were used. Membranes were
incubated with enhanced chemiluminescence substrate (Amersham ECL Prime Western
Blotting Detection Reagent, RPN2232, Cytiva, Marlborough, MA) following supplier’s
instructions and imaged with FUSION Solo S-Western Blot &Chemi Imaging system
(Vilber Lourmat, Eberhardzell, Germany).
COMET ASSAYS
Single-cell electrophoresis, or comet assays, were performed as previously described.28
Briefly, cells were gently harvested in the dark and embedded in low-melting-point
agarose. Then a thin layer of the cell suspension was added to slides previously coated
with normal-melting-point agarose. After solidification, slides were incubated for 1 hour
in cold lysis solution (10% DMSO, 1% Triton X-100) and 89% lysis buffer (NaCl 2.5 M,
EDTA 100 mM, Tris 10 mM, NaOH 200 mM, pH 10). Then electrophoresis was carried
out and the samples were fixed with absolute ethanol and stained with RedSafe 1:500
(21141, Intron Biotechnology, Seongnam, South Korea). Images were acquired with a Zeiss
