JCS037549 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. (a) Example of the progress of wound repair in wild-type (wt) and uPAR-deficient mice. uPAR knock-out (Ko) and wild-type (wt) skins were wounded and photographs were taken at the indicated time points. For each time point, wound diameters (distance between epithelial rims) were determined in at least five mice per genotype and the average wound length, expressed in mm, was plotted versus time after incision (b). P values underline the statistical significance of the difference between wt and uPAR Ko wound healing. Also see Table S1.

• Supplemental Figure S2 -

  Fig. S2. Macrophages and mast cells were counted in sections following immunohistochemistry or histochemistry for F4/80 or toluidine blue staining, respectively. At least three high-power fields immediately adjacent to the wound site were quantified and averaged. Also see Table S2.

• Supplemental Figure S3 -

  Fig. S3. Microvessel density was determined using immunofluorescence microscopy. Blood vessels were counted in CD31-labeled sections in a ×20 microscopic field centered on the wound site. Also see Table S3.

• Supplemental Figure S4 -

  Fig. S4. (a) uPAR-Ko keratinocytes with a murine or human uPAR expression vector, or an empty vector for control, were serum-starved for 18 hours and then stimulated with EGF (10 ng/ml) for 15 minutes. After protein extraction, cell lysates were subjected to immunoprecipitation with an antibody against EGFR followed by immunoblot analysis to detect phosphorylated EGFR (pTyr) and total EGFR. (b) uPAR-Ko keratinocytes with a human uPAR expression vector, or an empty vector for control, were tested for their growth rate in S-MEM 1% chelexed FCS in the absence (b) or in the presence of human uPA. Values are expressed as absorbance at 650 nm after staining with 0.1% crystal violet in 200 mM MES, pH 6.0, and the mean standard deviation (SD) of triplicate samples, over a 7-day period, is reported. (c) Suspended wild-type and uPAR-Ko keratinocytes were seeded on a glass surface (not shown) or on a ColIV-coated surface in the presence or in the absence of UO126 (10 µM). After 45 minutes, attached cells were detached with 10 mM EDTA, used for mRNA isolation and subjected to RT-PCR using LN5 (γ2 chain) primers (left panel), while secreted LN5 was detached from the surface with SDS-sample buffer, subjected to western blotting and probed for LN5 (γ2 chain) (right panel). Actin was used as a control. Results, expressed as amounts of secreted LN5 (γ2 chain)/actin (protein and mRNA) relative to levels in wild-type sample (=100%) ± SD, are representative of at least three experiments.

• Supplemental Table S1 -

  Table S1. The mean healing time, expressed in days ± standard deviations are shown. P values underline the statistical significance of the difference between wt and uPAR Ko wound healing.

• Supplemental Table S2 -

  Table S2. Mean number of macrophages and mast cells per field ± standard errors, at the indicated time points, are shown.

• Supplemental Table S3 -

  Table S3. Mean numbers of vessels per ×20 microscopic field ± standard errors, in at least 8 wounds per genotype for each time point, are shown.