JCS004481 Supplementary Material

Files in this Data Supplement:

• Supplemental Table S1 - Adobe PDF

• Supplemental Figure S1 -

  Fig. S1. Targeting strategy, and molecular analysis of recombinant ES cells and conditional plectin knockout mice. (A) Restriction map of the targeting construct, the 3′ end of the plectin locus, and the different plectin mutant alleles. Relevant exons are indicated as numbered black boxes, loxP sites as filled triangles, and FRT sites as empty triangles. The locations of probes, used to detect the various mutant alleles, are indicated. Restriction sites: E, EcoRI; H, HindIII; Nc, NcoI. (B) Southern blot analysis of recombinant ES cell clones. DNAs from targeted (lanes 2) and wild-type (Plec^+/+; lanes 1) ES cell clones were digested with EcoRI (left panel) or HindIII (right panel). EcoRI digestion produced fragments of 8.5 kb and 7.5 kb from the wild-type and mutant alleles, respectively; with HindIII, corresponding fragments of 29.5 kb and 17.5 kb were produced. Wild-type and mutant alleles were detected by hybridization of the filters with radiolabeled 5′ (left panel) and 3′ (right panel) mouse plectin genomic DNA probes. The targeted ES cell clones showed the mutated alleles (represented by shorter fragments), in addition to the wild-type alleles. (C) Southern blot analysis of genomic DNAs from genetically modified mice before (lane 1) and after (lane 2) deletion of the neo^r gene by FLP recombinase. DNAs from Plec^+/flox(neo)(lane 1) and Plec^{flox(neo)/flox(neo)} (lane 2) mice were digested with EcoRI and hybridized with the 5′ probe. Note the shorter fragment lacking neo (6.3 kb), generated from the Plec^flox(neo) allele (7.5 kb) after FLP-mediated recombination.

• Supplemental Figure S2 -

  Fig. S2. Immunofluorescence microscopy of frozen tracheal sections of Plec^floxf/lox (A,C) and K5-Cre KO (B,D) mouse specimens using anti-pan-plectin antiserum (A-D) and anti-K5,6,18 antibodies (C, D). Note, plectin expression is not reduced in the trachea of K5-Cre KO mice (A,B). Bar, 20 μm.

• Supplemental Figure S3 -

  Fig. S3. Relative expression levels of cytoskeletal and cell-cell adhesion proteins in detergent-soluble and -insoluble fractions from 1-day-old K5-Cre KO and Plec^flox/flox (control) skin. Proteins were extracted from skin as described in the text and equal amounts of proteins were analyzed by SDS-PAGE and immunoblotting using the indicated antibodies. (A) Coomassie brilliant blue staining. (B) Immunoblotting. Numbers underneath lanes represent the protein ratios relative to an arbitrary level of 1.0 determined by the control samples. One of two experiments is shown. Asterisks, bands used for quantification.

• Supplemental Figure S4 -

  Fig. S4. Intercellular junction formation in skin sections from 1-day-old Plec^flox/flox (control) and K5-Cre KO mice. Sections were immunolabeled using antibodies to E-cadherin (A,B), or to desmoplakin (E,F), or were labeled with TR-conjugated phalloidin to visualize F-actin (C,D). Note, (i) specific labeling of adherens junctions as a bright outline of cell-cell borders (A,B), (ii) similar actin network organization in control and K5-Cre KO skin (C,D), and (iii) uncompromised desmosome formation (E,F). Note also slightly reduced desmoplakin staining of suprabasal keratinocytes in K5-Cre KO epidermis (F). Dashed lines and asterisks in E and F indicate dermo-epidermal borders and hair follicles, respectively. Bar, 20 μm.

• Supplemental Figure S5 -

  Fig. S5. Immunolocalization of K10 (A,B) and involucrin (C,D) in the skin of 1-day-old Plec^flox/flox (control) and K5-Cre KO mice. Note similar K10 staining patterns in differentiating suprabasal keratinocytes (A,B) and indistinguishable involucrin patterns (C,D). Dashed lines, dermo-epidermal borders with invaginating hair follicles (asterisks). Bar, 20 μm.

• Supplemental Figure S6 -

  Fig. S6. Analysis of epidermal proliferation and keratinocyte survival. (A,B) Sections from the leg skin of 1-day-old Plec^flox/flox (control) and K5-Cre KO mice were subjected to immunoperoxidase staining using antibodies to Ki-67 and counterstained with Hematoxylin. (C) Statistical analysis of Ki-67-positive keratinocytes counted per linear millimeter of interfollicular epidermis basement membrane. Mean values ± standard deviations of three littermate pairs are shown. Note that the numbers of Ki-67-positive cells were similar in control and K5-Cre KO epidermis. (D,E) Control and K5-Cre KO skin sections were subjected to TUNEL assays to detect the presence of apoptotic cells. Nuclei were stained with Hoechst. Arrows indicate apoptotic cells in the suprabasal layers of the epidermis. No signs of accelerated cell death of basal keratinocytes were visible in interfollicular K5-Cre KO epidermis. The staining along the surface of the stratum corneum is non-specific. Bars, 20 μm.