D4693

To analyze cell cycle, keratinocytes were isolated from adult mice tail skin and P0 neonate skin using dispase II digestion (Sigma, catalog D4693, 2 mg/mL) at 4°C overnight. Isolated keratinocytes were fixed and permeabilized using Cytofix/Cytoperm Fixation/Permeabilization Solution Kit (BD Biosciences, catalog 554714). Permeabilized keratinocytes were stained with DAPI (10 μg/mL) in Perm/Wash buffer (BD Biosciences) for 30 minutes at 4°C in the dark and analyzed on an LSRII flow cytometer (BD Biosciences) or CytoFLEX (Beckman Coulter) and data were analyzed using FlowJo software. For protein synthesis measurement in vivo, mice were injected intraperitoneally with OP-Puro (Medchem Source, catalog JA-1024) (50 mg/kg body weight, pH 6.4–6.6 in PBS). Mice were euthanized 1 hour later, and dorsal skin was collected. Keratinocytes were isolated as described above. Fluorescence labelling of OP-Puro was performed using Click-iT Plus OPP Alexa Fluor 488 Protein Synthesis Assay Kit per the manufacturer’s instructions (Thermo Fisher Scientific, catalog C10456). Mice injected with PBS were used as control to determine background labelling. Labelled cells were analyzed on LSRII flow cytometer (BD Biosciences) or CytoFLEX (Beckman Coulter). Flow cytometry data were analyzed using FlowJo software.

For the isolation of BMDMs, bone marrow cells were flushed from hindlimbs of 6‐ to 8‐week‐old mice with α‐MEM (Gibco, Thermo Fisher Scientific) supplemented with 10% FBS (Gibco, Thermo Fisher Scientific) and 1% penicillin‐streptomycin (Gibco, Thermo Fisher Scientific). The cells were pelleted and seeded in a 10‐cm dish at 37 °C in a 5% CO₂ humidified incubator overnight. After overnight incubation, the adherent cells were removed, and the non‐adherent cells were collected for BMDM differentiation. The non‐adherent cells were again incubated in the medium with 30 ng mL⁻¹ mouse M‐CSF (macrophage‐colony stimulating factor) (Novoprotein, Summit, NJ) to 80% confluence and reseeded for mechanical compression experiments.
To isolate the WPCs, procedures were adapted from a previous study.^[43] Briefly, hindlimbs were dissected from 6‐ to 8‐week‐old mice. After careful removal of muscle fibers and tendons, the harvested tissue was digested for 10 min at 37 °C with 3 mg mL⁻¹ collagenase (C0130, Sigma‐Aldrich) and 4 mg mL⁻¹ dispase (D4693, Sigma‐Aldrich). After the digestion, the supernatant was discarded, and the tissue was further digested for an additional 50 min. The released cells were plated on a collagen I‐coated 10‐cm dish and cultured in the medium with 30 ng mL⁻¹ mouse M‐CSF for 3 d. The adherent cells were then collected for mechanical compression experiments.

Skin samples were incubated with 400 μl PBS (0.35% dispase II, D4693, Sigma-Aldrich, USA) at 4 ℃ for 3 h. The sample was then wash, cut, and incubated with 200 μl PBS (1% collagenase, C0130, Sigma-Aldrich, USA) at 37 ℃ for 4 min and was filtered with 70-micron screen and centrifuged at 1500 g for 5 min. Wash and incubated with F4/80 antibody (123,110, Biolegend, San Diego, CA, USA) at 4 ℃ for 30 min. Wash and centrifuged at 1500 × g for 5 min. Filtration with 40-micron screen and measured by fluorescence-activated cell sorting (FACS, C6 Plus, BD, New York, USA).

Tissues were fixed in 4% paraformaldehyde, processed, and embedded in paraffin. Skin thickness was defined as the distance between the granular layer of the epidermis and the panniculus carnosus muscle. Immunolabeling experiments were performed on slides that were citrate buffer–treated for heat-mediated antigen retrieval. Sections were permeabilized in 0.5% Triton X-100 and blocked with serum in which the secondary antibodies were raised. Primary antibodies are listed in Supplemental Table 1. DAPI was used to stain nuclei (D3571, Thermo Fisher Scientific). Secondary Alexa Fluor 488/555/647 antibodies were used at a dilution of 1:200 (Thermo Fisher Scientific). FITC-conjugated Griffonia Simplicifolia Lectin I isolectin B4 (FL-1201, Vector Laboratories) was used at a dilution of 1:100. Hind paws were treated with dispase II (20 mg/mL) (D4693, Sigma-Aldrich) overnight and stained with Oil Red O to assess sebaceous glands and with Nile Blue to stain sweat ducts.