Integrin 3β1 inhibits directional migration and wound re-epithelialization in the skin

JCS029108 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Targeting strategy and generation of epidermis-specific Itga3-knockout mice. (A) Restriction maps of the 5′ end of the Itga3 locus, the targeting construct, and the different Itga3 alleles. Exons are represented as numbered boxes, with black boxes indicating coding regions and gray boxes indicating non-coding regions. Triangles mark loxP sites. Position of the unique HpaI and BamH1 sites used for insertion of the single loxP and the floxed neo-tk cassette are indicated, as are the primers used to detect the different alleles of Itga3. (B) Southern blot of recombinant embryonic stem cell clones. DNA from independently targeted clones or wild-type embryonic stem cells was digested with either BamHI or EcoRI, resolved by agarose gel electrophoresis, and blotted onto nitrocellulose membranes. Wild-type and mutant alleles were detected by hybridization with radiolabeled probes corresponding to exon 2 and exon 3 (3′ probe) and exon 1 (5′ probe). Random recombination is indicated with an asterisk. (C) PCR analysis of genomic DNA of conditional Itga3 mice before and after crossing with K14-Cre-mice. The floxed Itga3 allele was detected in tail DNA using primers P1 and P2, the K14-Cre transgene was detected using primers K14-Cre3 and K14-Cre5, and the null allele was detected with primer pair P1 and P3. PCR products were resolved by agarose gel electrophoresis and visualized with ethidium bromide. The size of DNA fragments is indicated in bp.

• Supplemental Figure S2 -

  Fig. S2. Distribution of integrins and BM proteins in the migrating epidermis. (A) Schematic representation of the leading edge of the epidermis migrating into a wound bed (top left), depicting areas of: (a) Ln-332 deposition and upregulation of integrins, and (b) deposition of BM proteins Nd, Ln-511, and Col-IV. A cryosection of a 3-day-old wound inflicted in an Itga3^flox/flox mouse was stained with antibodies against integrin α3 and Ln-511 (top right). A cryosection of a 3-day-old wound from an Itga3^flox/flox mouse was stained with antibodies against integrin β1 and Nd (bottom). Scale bars: 100 μm. (B) Cryosections of 3-day-old wounds from Itga3^flox/flox (upper panel) or Itga3^flox/flox; K14-Cre mice (lower panel) were stained with antibodies against integrin β1, integrin α5, integrin α6 and integrin β4, as well as the BM proteins Col-IV, Ln-511, Nd and Ln-332. The granulation tissue may be lost as a consequence of sectioning. Scale bar: 150 μm.

• Supplemental Figure S3 -

  Fig. S3. Characterization of keratinocyte cell lines MKα3(+) and MKα3(−). (A) FACS analysis of the expression levels of α3 at the surface of MKα3(+) and MKα3(−) cells (upper panel). Bottom panel shows immunoblots demonstrating the expression of keratinocyte and epithelial markers in MKα3(+) and MKα3(−) cells. Murine mammary cell line Rac-11P and murine fibroblast cell line NIH3T3 were included as a positive and negative control, respectively. (B) Proliferating MKα3(+) and MKα3(−) cells were incubated for 2 hours with 10 μM BrdU, and the number of BrdU(+) nuclei was determined by immunofluorescence. Proliferation in MKα3(+) cells was set to 100%. In each experiment ∼200 cells were scored, and the values shown are the means ± s.e.m. of three independent experiments. (C) MKα3(+) and MKα3(−) were maintained in high Ca²⁺ for 18 hours and then fixed and stained for occludin, ZO-1, E-cadherin and β-catenin. Nuclei were counterstained with DAPI. Scale bar: 20 μm. (D) MKα3(+) and MKα3(−) were maintained in high Ca²⁺ for 48 hours, fixed, and stained for plectin and integrin β4 to determine HD assembly. Scale bar: 20 μm.

• Supplemental Figure S4 -

  Fig. S4. Adhesion and cell spreading in primary keratinocytes. Primary keratinocytes were isolated from neonatal Itga3^flox/flox and Itga3^flox/flox; K14-Cre mice. (A) Cells were seeded onto Col-1 or Ln-332 in K-SFM without supplements in the absence or the presence of α6-blocking antibody GoH3 (10 μg/ml). After 30 minutes, non-adherent cells were washed away. Values shown represent the average percentages of adherent cells from two experiments performed in triplicate (*P<0.05). (B) Cells were seeded on Col-1 or Ln-332, allowed to spread, and then incubated with α6-blocking antibody GoH3 (10 μg/ml). After 3 hours, the number of spread cells was scored and expressed as a percentage of the total number of cells. The graphs depict the averages of two experiments. In each independent experiment, approximately 500 cells per condition were counted (*P<0.05, ***P<0.0005).