**4. Material and Methods**

#### *4.1. Animal Models and Wound Area Analysis*

C57BL/6J mice were purchased from Jackson Laboratory. SphK1 KO and S1PR2 KO mice were from Dr. Richard Proia (The National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) of National Institutes of Health (NIH)). All animal procedures were approved by the Institutional Animal Care and Use Committee of the Virginia Commonwealth University on 1 July 2015 (AD20100) and the Animal Experimental Ethical Review Committee of Nippon Medical School on 7 September 2016 (28-054). The murine excisional wound splinting model was generated as described previously [25]. Mice were anesthetized with isoflurane, and their dorsal hair was removed. Two 5 mm-diameter full-thickness skin punches were generated symmetrically on either side of the midline. Circular 12 mm-diameter silicon rubber splints were punched in the center to generate 6 mm-diameter holes. They were then fixed with instant-bonding adhesive and sutures around the punch wounds. After applying the required ointment, the wounds were dressed with Tegaderm (3M, Maplewood, MN, USA). The wounds were photographed at the indicated time points. The digital photos were analyzed using GIMP 2.8 software. The pixels of the wound area were normalized to the pixels of the inside of the silicon splint. The wound areas at the indicated time points were expressed as ratios relative to the wound area immediately after wounding.

## *4.2. Cells and S1P Preparation*

Murine dermal fibroblast NIH3T3 cells, human cervix epithelioid carcinoma HeLa cells, and human embryo kidney HEK293 were cultured in Dulbecco's modified eagle medium (DMEM) containing 10% fetal bovine serum (FBS). To analyze collagen production, ascorbic acid 2-phosphate (Sigma-Aldrich, Carlsbad, CA, USA) was added to the culture medium to a concentration of 0.2 mM. S1P was purchased from Sigma-Aldrich and 1 mM was sonicated in 4% bovine serum albumin (BSA). Recombinant human TGFβ-1 was purchased from R&D systems (Minneapolis, MN, USA). VPC23019 was purchased from Avanti (Alabaster, AL, USA). JTE013 was purchased from Cayman Chemical (Ann Arbor, MI, USA).

#### *4.3. Plasmid Construction, In Vitro Transfection with sCA-Encapsulated Plasmids, and Preparation of Plasmid-sCA Ointment*

The murine SphK1 gene was amplified using TaKaRa Ex Taq Hot Start Version (TaKaRa, Kusatsu, Japan). The SphK1-expressing plasmid was then constructed using the pcDNA3.1/V5-His TOPO TA Expression Kit (Invitrogen, Carlsbad, CA, USA). sCA-encapsulated plasmids were prepared as described previously [27,47]. Thus, 4 μL of 1 M CaCl2 was incubated at 37 ◦C for 30 min with 2 μg of plasmid DNA in 1 mL of an inorganic solution (NaHCO3, 44 mM; NaH2PO4, 0.9 mM; CaCl2, 1.8 mM; pH 7.5) and then centrifuged at 12,000 rpm for 3 min. After the pellet was dissolved with DMEM, the solution was sonicated in a water bath for 10 min, thus generating sCA-encapsulated plasmids. Cells cultured in 6-well plates for 24 h were incubated with the plasmid-sCA-DMEM solution for 6 h. The medium was then replaced with DMEM containing 10% FBS. After another 48 h, the cells were collected for total RNA isolation. The plasmid-sCA ointment was generated by dissolving the sCA pellet with 50 μL of PBS, mixing it with 100 μg of plasmid DNA, and then mixing the solution into 200 μL of Aquaphor ®. The four wounds of two mice were each treated once with 250 μL of ointment.

#### *4.4. Sponge Granulomas in Mice and Their Injection with Plasmid-sCA*

Sponge granulomas were generated in two mice as described previously [32]. Thus, polyvinyl alcohol (PVA) sponges were processed to generate 10 mm-diameter 3 mm-thick sponges. The sponges were irradiated with ultraviolet light and infiltrated with saline for 48 h. The sponges were then transplanted (two per mouse) in the subcutaneous dorsal area in a symmetrical fashion. sCA was mixed with 50 μg of plasmid DNA and pelleted. The pellet was then dissolved in 100 μL of saline containing 0.5% mouse serum albumin. The two sponges on one mouse were injected every other day with 50 μL of sCA-plasmid. The two sponges on the other mouse were injected with sCA-vector. The samples were harvested 14 days after transplantation and subjected to histological analysis. The percentage of total area that was occupied by eosin-positive area in the sponges were analyzed by using ImageJ. Four fields per sponge were analyzed and the averages were calculated.
