Ethics statement

The animal study protocol was reviewed and approved by the Animal Experiment Ethics Committee of Sookmyung Women’s University, Republic of Korea (Approval number: SMWU-IACUC-1609-027). All experiments were performed according to approved guidelines.

Peptide synthesis

AES16-2M (REGRT) and scrambled control peptide were synthesized at PEPTRON (Republic of Korea) in the form of lyophilized powder (1 mg/vial) and analysed by high-performance liquid chromatography (HPLC) analysis to confirm a purity of >98%. The molecular weight of AES16-2M was 618 Da as determined by mass spectrum analysis.

Cell culture

HaCaT cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Welgene, Republic of Korea) containing 10% heat-inactivated foetal bovine serum (FBS, Welgene), 100 U/ml penicillin, and 100 µg/ml streptomycin (Gibco, CA, USA). HDF were obtained from ATCC and cultured in the recommended growth media (ATCC, VA, USA). Cells were grown and maintained at 37 °C in a humidified incubator at 5% CO 2 .

Mice wound healing experiments

Seven-week-old BALB/c wild-type and BALB/c-nude female mice were purchased from Orient (Republic of Korea) and maintained for 1 week prior to initializing the experiments. In order to create acute wounds, the dorsal skin was shaved, the mice anesthetized with avertin, and a 10-mm-diameter full-thickness wound was punched onto the back using a biopsy punch. Phosphate-buffered saline (PBS), AES16-2M (0.5 μg/wound), or EGF (10 μg/wound, Prospec, Israel) in 50 μl of 20% Pluronic® F-127 gel (Sigma, MO, USA) was applied to the wound area five times. The wound area was photographed and measured by digital planimetry using ImageJ software (National Institutes of Health, MD, USA).

Cell scratch wounding assay

HaCaT cells (3 × 105 cells/well) were seeded in 12-well plates and cultured as a monolayer to confluence in serum-conditioned medium. After overnight, cells were incubated in serum-free media containing 10 μg/ml mitomycin C (Sigma) for 2 hr to completely inhibit cell proliferation. Scratch wounds were created in confluent monolayers using a P200 disposable micropipette tip. After the suspended cells were removed by washing with serum-free medium, the wounded monolayers were cultured in complete medium with or without AES16-2M. For the inhibitor treatment group, cells were pre-treated with 0.1 µM U0126 (Calbiochem, Germany) 1 hr prior to AES16-2M treatment. Wound area recovery was observed under a phase-contrast microscope and photographed. Using the ImageJ program, the size of the opened area was measured from the digital images. Six randomly selected images were acquired for each group. All experiments were independently carried out in triplicate.

Transwell migration assay

The fibroblast migration assay was performed using a transwell chamber (Costar, MA, USA) with 6.5-mm-diameter and 8.0 µm-pore polycarbonate filters. Prior to performing the migration assay, cells were treated with or without AES16-2M overnight and then harvested. The lower chamber was filled with 600 μl DMEM supplemented with 10% FBS, and the cells were seeded at a density of 1 × 104 cells in 100 μl serum-free medium in the upper chamber followed by incubation at 37 °C. After 24 hr, the transwell inserts were fixed with methanol, stained with 0.5% crystal violet in 10% ethanol for 10 min, and washed with distilled water (DW). Cells in the upper compartment were removed using a cotton swab and then photographed using light microscopy. For elution, the dyed transwell inserts were dissolved with 10% acetic acid for 20 min and analysed with an enzyme-linked immunosorbent assay (ELISA) microplate reader (Molecular Devices, CA, USA) at 570 nm.

Immunohistochemistry

Wounded areas surrounded by unwounded skin were dissected on day 4 after injury, fixed in 4% paraformaldehyde, and embedded in paraffin. After slicing into sections, slides were deparaffinized, rehydrated, and H&E staining performed using standard techniques. For immunohistochemistry, antigen retrieval was performed by microwave treatment of the slides for 10 min in citrate buffer (pH 6.0) (Abcam, Cambridge, UK). Slides were blocked with 5% horse serum (Vector Laboratories, CA, USA) in PBS at room temperature for 1 hr and then incubated with rabbit anti-CD31 (1:50; ab28364, Abcam), anti-keratin 14 (1:200; ab181595, Abcam), and anti-phosphorylated (p-)ERK 1/2 (1:100; 4376, Cell Signaling, MA, USA) at 4 °C overnight. After washing, slides were incubated for 30 min with horseradish peroxidase (HRP)-conjugated anti-rabbit antibodies (7074, Cell Signaling) and visualized with 3,3′-diaminobenzidine (DAB) staining (Abcam). Counter-staining was performed with haematoxylin solution (Dako, CA, USA) and slides were then dehydrated and mounted.

Morphometric analysis of wounds

Morphometric analysis was performed on digital images using the ImageJ program. The extent of re-epithelialization and granulation tissue formation was determined using H&E- and anti-keratin 14 antibody-stained paraffin tissue sections. The length of the epithelial tongue was determined as the distance between the epithelial tip and the margin of the wound. The leading-edge ratio was calculated as the ratio of the length of wound to the length of the epithelial tongues by referring to previous research37. Granulation tissue is defined as the tissue matrix, which includes a variety of cell types such as ECM, immune cells, vascular tissue, and fibroblasts14. The granulation tissue area was defined as between the wound margins, underneath the neo-epithelium, and above the subcutaneous fat tissue according to previous research27.

Western blot

HaCaT cells were washed twice with PBS and lysed using Pro-prep protein extraction solution supplemented with protease inhibitor mixtures (Intron Biotechnology, Republic of Korea) on ice. Protein extracts were separated by 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad, CA, USA). The membrane was blocked with 5% non-fat dry milk in Tris-based saline with Tween-20 (TBS-T) for 1 hr and then incubated with rabbit anti-ERK 1/2, anti-p-ERK 1/2 (all at 1:1000; 4695, 9101, Cell Signaling) and anti-GAPDH (1:2000; sc-25778, Santa Cruz, MA, USA) overnight at 4 °C. After washing, the membranes were incubated for 1 hr with appropriate HRP-conjugated secondary antibodies (1:2000; 7074, Cell Signaling) at room temperature for 1 hr. The protein-antibody complexes were detected using an ECL reagent on the ChemiDoc imaging system (Fuji, Japan). The images were quantified with the ImageJ program.

Statistical analysis

All experiments were performed independently at least three times. Data are expressed as means ± SD. The comparisons between the control and treated groups were ascertained using a two-tailed student t test and comparisons among groups were determined using one-way analysis of variance (ANOVA) followed by Tukey post-hoc test. A p value < 0.05 was considered statistically significant. Statistical analysis was performed using GraphPad PriSM version 5 for Windows (GraphPad Software, CA, USA).