Fig 1.
Effect of all-L and all-D Esc(1-21) peptides at different concentrations on the number of metabolically-active HaCaT cells after a short (A) or long (B) term treatment.
Cells were plated in wells of a microtiter plate, at 4x104 cells/well. After overnight incubation at 37°C in a 5% CO2 atmosphere, the medium was replaced with 100 μl fresh medium supplemented with the peptides at different concentrations. After 2h (A) or 24h (B) of peptide treatment, cell viability was determined by the MTT reduction to insoluble formazan (see Materials and Methods for additional information). Cell viability is expressed as percentage with respect to the vehicle-treated control cells. Data points represent the mean of triplicate samples ± SEM. The level of statistical significance between all-L and all-D peptides is indicated as follows: ***, p<0.001.
Fig 2.
Effect of all-L and all-D Esc(1-21) on the closure of a pseudo-"wound" field produced in a monolayer of HaCaT cells.
HaCaT cells were seeded in each side of an ibidi culture insert and grown to confluence. Afterwards, they were treated with all-L (A) or all-D (C) peptide at different concentrations, as indicated. Cells were photographed at the time of insert removal (0 h) and examined for cell migration after 3, 6, 9 and 12h from peptide addition. The percentage of cell-covered area at each time point is reported on the y-axis. Control (Ctrl) is given by vehicle-treated cells. All data are the mean of three independent experiments ± SEM. The levels of statistical significance between Ctrl and peptide-treated samples are indicated as follows: *, p<0.05; **, p<0.01;***, p<0.001. (B): micrographs showing representative results of pseudo-"wound" closure induced upon treatment of keratinocytes with 0.25 μM all-L Esc(1-21) or all-D Esc(1-21) with respect to the Ctrl sample.
Fig 3.
Effect of all-L Esc(1-21) on the closure of a pseudo-"wound" field produced in a monolayer of primary human keratinocytes.
(A): two monolayers of primary keratinocytes separated by a defined distance were treated with the peptide at different concentrations, as indicated. Cells were photographed at the time of insert removal (0 h) and examined for cell migration after 15, 20 and 24h from peptide addition. The percentage of cell-covered area at each time point is reported on the y-axis. Control (Ctrl) is given by vehicle-treated cells. All data are the mean of three independent experiments ± SEM. The levels of statistical significance between Ctrl and peptide-treated samples are indicated as follows: *, p<0.5; ***, p<0.001. (B): micrographs showing representative results upon treatment of primary keratinocytes with all-L Esc(1-21) (4 μM and 10 μM) with respect to the Ctrl sample. The black line marks off the cell-free area in the peptide-treated samples after 15 and 20 h. No cell-free area was noted in samples treated with 10 μM peptide after 20 and 24h.
Fig 4.
Effect of LL-37 on the closure of a pseudo-"wound" field produced in a monolayer of HaCaT cells.
(A): two monolayers of HaCaT cells separated by a defined distance were treated with LL-37 at different concentrations, as indicated. Cells were photographed at the time of insert removal (0 h) and examined for cell migration after 3, 6, 9 and 12h from peptide addition. The percentage of cell-covered area at each time point is reported on the y-axis. Control (Ctrl) is given by vehicle-treated cells. All data are the mean of three independent experiments ± SEM. The level of statistical significance between Ctrl and peptide-treated samples is indicated as follows: ***, p<0.001. (B): micrographs showing representative results upon treatment of keratinocytes with LL-37 (0.25 μ M, 1 μ M and 4 μ M) with respect to the Ctrl sample.
Fig 5.
Effect of mitomycin C (Mito) on the peptides-mediated closure of a pseudo-"wound" field produced in a HaCaT cells monolayer.
(A) After removal of the ibidi culture insert, HaCaT cell monolayers were pre-incubated or not with 3 μM Mito for 30 min and subsequently treated with 0.25 μM all-L Esc(1-21) or LL-37. Cells incubated with medium served as control (Ctrl). Samples were photographed at different time intervals, as indicated in the legends to Figs 2 and 4, and the percentage of cell-covered area was calculated and reported on the y-axis. All data are the mean of three independent experiments ± SEM. No statistical significance was found between samples treated with the peptide alone or pre-incubated with Mito.
Fig 6.
Effect of all-L Esc(1-21) on the phenotype of HaCaT cells.
Cells (150,000) were seeded on a glass coverslip and treated or not with 0.25 μM peptide in DMEMg for 12h at 37°C and 5% CO2. Afterwards, samples were washed three times with PBS and fixed with 3.7% formaldehyde for 10 min at +4°C. Then, they were permeabilized with 0.1% Triton X-100 for 10 min at room temperature and stained with phalloidin-fluorescein isothiocyanate (40 μM in PBS) for 30 min at room temperature, to visualize the cytoskeleton. The nuclei were stained by adding 50 μl of Hoechst 33258 (2 μg/ml) for 10 min at room temperature. The coverslips were mounted on slides and observed under a fluorescent microscope (KOZO OPTICS XJF800) at 40 x magnification and photographed with a Color View II digital camera. (A): vehicle-treated control cells; (B) peptide-treated cells. Insets represent the magnification of the image portion indicated by the white frame. rows indicate filopodial formation.
Fig 7.
Effect of AG1478 and GM6001 inhibitors on the all-L Esc(1-21)-mediated closure of a pseudo-"wound" field produced in a HaCaT cells monolayer.
After removal of the ibidi culture insert, HaCaT cell monolayers were pre-incubated with 0.2 μM AG1478 for 15 min or with 25 μM GM6001 for 30 min and subsequently treated with 0.25 μM all-L Esc(1-21). Cells incubated with medium served as control (Ctrl). Samples were photographed at different time intervals, as indicated in the legend to Fig 2, and the percentage of cell-covered area was calculated and reported on the y-axis. All data are the mean of three independent experiments ± SEM. The levels of statistical significance between inhibitor-pretreated groups and samples treated with the peptide alone are indicated as follows:*, p<0.05; **, p< 0.01; ***, p< 0.001.
Fig 8.
Phosphorylation of STAT3 by all-L Esc(1-21).
About 1 x 106 keratinocytes were stimulated with 0.25 μM all-L Esc(1-21) or LL-37 for 20 min. Control (Ctrl) was given by vehicle-treated cells. Cells were harvested into lysis buffer and phospho-STAT3 (Tyr 705) was evaluated by performing an ELISA assay (see Materials and methods). All data are the mean of three independent experiments ± SEM. The level of statistical significance between peptide-treated samples and Ctrl is indicated as follows: ***, p < 0.001.
Fig 9.
Images of HaCaT cells treated with rhodamine-labeled all-L Esc(1-21) at different times (30 min and 24h).
Panels A and A' show bright field images. Black arrows indicate keratinocytes. B and B' show the Hoechst fluorescence signal which indicates the nuclei position. Panels C and C' show the distribution of the rhodamine-labeled Esc(1-21). Red fluorescence is not detectable at the level of nuclei (white arrows), but rather in the cytoplasm and mainly concentrated at the nuclei periphery, already after a short incubation time (C). Panels D and D' show the overlay of the two fluorescent probes. All images are z section images taken from the mid-cell height. All bars indicate 10 μm.