450 JOURNAL OF COSMETIC SCIENCE
medium, glucose, and palmitic acid were used as a metabolic stress control. After 48 hours
of incubation with treatments, a couple of washes were made with PBS (phosphate-buffered
saline, Sigma-Aldrich, St. Louis, MO, USA), and microscopic images and measurements
were performed with a high-content screening system (Operetta® High-Content Imaging
System, PerkinElmer, Inc., Waltham, MA, USA) and processed using Harmony® software
(PerkinElmer, Inc., Waltham, MA, USA). Fluorescence intensities were measured using a
Hoechst33342 channel (Emission: 410-480 nm) and were corrected using the cell number
determined by DPC (Digital Phase Contrast). These values were normalized by the control
or metabolic stress control when applicable. Three independent assays were performed in
four wells per condition. Statistical significance was calculated using an unpaired student’s
t test.
INHIBITION OF IL1ALPHA RELEASE IN HUMAN EPIDERMAL KERATINOCYTES
HEKa cells were seeded and incubated in a culture medium following the same protocol
described in the previous experiment (Inhibition of HMGB1 stressorin release). Cells
treated with the culture medium, glucose, and palmitic acid were used as a metabolic
stress control. Cells treated with the culture medium alone were used as a control. Cells
treated with a specific RAGE-blocking antibody (ABCAM) were used as a control for
IL1alpha release inhibition. After 24 hours of incubation, the supernatants were collected,
and a protease inhibitor cocktail (Merk Life Science) was added they were then kept at
-80°C until the supernatants were analyzed to determine the IL1alpha level by AlphaLISA
(PerkinElmer, Inc., Waltham, MA, USA) according to the manufactureŕs protocol. These
results were normalized with the total viable cell number for each condition calculated
by the PrestoBlueTM staining assay (Thermo Fisher Scientific, Waltham, MA, USA). The
obtained values were normalized versus the control or metabolic stress control as applicable.
Each condition was tested in three to four independent experiments and in biological
triplicates. Statistical significance was calculated using an unpaired student’s t test.
QUANTIFICATION OF MBNL1 PROTEIN LEVELS ON HUMAN SKIN FIBROBLASTS
Electrical stimulation of fibroblasts was performed by seeding cells in 12-well plates over
sterile glass slides and incubating them at 37°C and 5% CO
2 for 48 hours. The glass
slides were transferred to 6-well plates and were electro-stimulated (1.5 V, 70 mV/mm,
1000 µA) for 1 hour using a C-dish device (IonOptix, Westwood, MA, USA) at room
temperature. An identical 6-well plate with nonelectrostimulated cells were incubated
at room temperature for 1 hour (control). After electro-stimulation, glass slides were
transferred again to a 12-well plate and incubated at 37°C and 5% CO
2 for 24 hours. Three
different replicates were done for each condition. Tetrapeptide-1 treatment was performed
by seeding cells in 96-well plates and incubating them at 37°C and 5%CO
2 for 48 hours.
After the incubation time, cells were treated with tetrapeptide-1 at 0.01 mg/mL, 0.1 mg/
mL, and 0.5 mg/mL, respectively, for 24 hours. Cells incubated with the medium alone were
used as a control. Three different replicates were done for each condition. After 24 hours
of incubation, cells were washed twice with PBS, fixed with 4% PFA (paraformaldehyde
solution) for 15 minutes, and washed twice more. After that, cells were permeabilized with
1% X-triton solution for 15 minutes and blocked with a 5% BSA (bovine serum albumin)
solution for 1 hour. Cells were then incubated with a 1/20 dilution of the primary antibody

451 DELIVERING SUSTAINABLE SOLUTIONS TO IMPROVE WELLBEING
against MBNL1 protein (Sigma-Aldrich, St. Louis, MO, USA) in a 5% BSA solution for 2
hours. After the incubation time, cells were washed and incubated with a 1/200 dilution of
the secondary antibody (Alex Fluor 488) in a 5% BSA solution for 1 hour. Finally, cells were
washed three times and stained with a DAPI mount solution for nuclei detection. MBNL1
protein quantification was measured as fluorescence intensity of MBNL1 normalized by
the number of nuclei for each condition using a confocal microscope (Operetta® confocal
microscope, PerkinElmer, Inc., Waltham, MA, USA) with the Alexa Fluor 488 Green
channel (Ex is 460-490 nm/Em is 500-550 nm). Results are expressed as a variation of
relative MBNL1 protein levels (%)with respect to the control condition (without electrical
stimulation) and the electrical stimulated condition.
DETECTION OF EDA-FIBRONECTIN MARKER ON HUMAN DERMAL FIBROBLASTS
Cells coming from a collagen-gel contraction assay (see the following section) were used
to detect an EDA-fibronectin marker. The cells included in that assay were the control
condition (without electrical stimulation), electrical stimulated cells, and tetrapeptide-1
treated cells at 0.01 mg/mL. 3D collagen gels were previously digested using a collagenase
treatment and were fixed on 12-well plates into coverslips. The cells were then washed
twice with PBS (Sigma-Aldrich, St. Louis, MO, USA) and permeabilized with 0.5% Triton
X-100 (Sigma-Aldrich, St. Louis, MO, USA)) for 10 minutes. Finally, cells were washed
three times with PBS again prior to fluorescent labeling of the target protein. Samples were
blocked with 4% BSA (Sigma-Aldrich, St. Louis, MO, USA)) in PBS for 2 hours. After this
step, cells were incubated with an antifibronectin [IST-9] mouse monoclonal antibody at
1:500 (ab6328) (ABCAM) and diluted in 4% BSA in PBS for 2 hours. After the incubation
time, cells were washed again with PBS three times and secondary Alexa Fluor® Thermo
Fisher Scientific, Waltham, MA, USA) 488 goat antimouse IgG (H +L) antibody (1:500
Life Technologies, green fluorescence emission dye) was added and cells were incubated
for 1 hour in the dark. After three washes with PBS, the nuclei of the cells were stained,
and coverslips were mounted with a ProLong Gold antifade reagent with DAPI (Life
Technologies, Carlsbad, CA, USA). The fluorescence intensity signal was determined using
an Operetta® confocal microscope (PerkinElmer, Inc, Waltham, MA, USA) using Alexa
Fluor 488 Green channel (Ex is 460-490 nm/Em is 500-550 nm) and was corrected by the
determining the number of nuclei for each condition. Four independent experiments were
analyzed.
IN VITRO COLLAGEN-GEL CONTRACTION ASSAY
Human dermal fibroblasts were seeded in 60 mm Petri dishes prepared for the electrical
stimulation. After an overnight period to allow cell attachment in the conditions of
treatment, tetrapeptide-1 was added to the culture media at a concentration of 0.01 mg/
mL for 24 hours (in duplicate experiments). The peptide was also maintained in the
culture media for the 3D cultures thus, the total treatment time was 48 hours. Control
experiments (with and without electrical stimulation) were performed in the same way
but without adding the tetrapeptide-1. Therefore, there were three groups in the study:
control without electrical stimulation, control with 1 hour of electrical stimulation, and
the peptide treatment (tetrapeptide-1 at 0.01 mg/mL). Cell suspensions for each of the
conditions to seed were diluted in each corresponding medium with the peptide. The