Article Figures & Data
Figures
- Figure 1. Binding of recombinant laminin-111, laminin-511, and their EQ mutants to integrins.
(A) Schematic diagram of a full-length laminin. The Glu (E) residue in the C-terminal region of the γ1 chain critical for integrin binding is indicated. (B–L) Binding of recombinant integrin α1β1 (B), α2β1 (C), α3β1 (D), α6β1 (E), α6β4 (F), α7x1β1 (G), α7x2β1 (H), α9β1 (I), αvβ3 (J), αvβ5 (K), and α5β1 (L) to immobilized laminins (LMs), type-I and type-IV collagens, polydom, and vitronectin. (M) Anti-FLAG antibody binding for quantification of immobilized recombinant proteins. Vertical axes represent absorbance at 490 nm which indicates integrin binding. Data represent means ± SD of triplicate assays.
- Figure S1. Recombinant laminins used for in vitro binding assay.
(A) Coomassie Brilliant Blue staining of affinity-purified recombinant laminin-111, laminin-511, and their EQ mutants. SDS–PAGE was carried out in 4% polyacrylamide gels under reducing conditions. (B) Western blot analyses of purified recombinant laminin-111, laminin-511, and their EQ mutants with mAbs against α1, α5, β1, and γ1 subunits to confirm the authenticity of the purified laminin isoforms.
- Figure S2. Binding of recombinant laminin-111, laminin-511, and their EQ mutants to integrins.
(A) Binding of recombinant integrin α5β1 to immobilized laminins, type I and IV collagens, polydom, vitronectin, and fibronectin included as a positive control ligand for integrin α5β1 to verify recombinant integrin α5β1 activity. (B) Binding of recombinant integrin α6β1 to immobilized human laminin-111 (LM111), its EQ mutant, and mouse laminin-111 purified from Engelbreth-Holm-Swarm tumor (EHS-LM) under increasing coating concentrations (5, 25, and 100 nM). Vertical axes represent absorbance at 490 nm, which indicates integrin binding. Data represent means ± SD of triplicate assays.
- Figure 2. Generation of Lamc1EQ/EQ knock-in mice.
(A) Schematic diagram of the targeted mutation of Lamc1. The open boxes represent exons. The knock-in construct was designed to replace WT exon 28 encoding Glu at residue 1,605 with a mutant exon 28 encoding Gln at residue 1,605. The probes used for Southern blotting are indicated by bold lines. (B) Southern blot analyses of genomic DNA from WT, Lamc1EQ(neo)/+, and Lamc1EQ/+ offspring after digestion with SexAI and PacI. The detection of 9.0- and 7.4-kbp fragments with probe 1 and probe 2, respectively, in the Lamc1EQ(neo)/+ lanes indicates the occurrence of the expected homologous recombination. The detection of a 7.4-kbp fragment with probe 1 in the Lamc1EQ/+ lane indicates that the neomycin-resistance gene has been removed from the Lamc1EQ(neo) allele by the Cre-loxP system. (C) Survival of WT, Lamc1EQ/+, and Lamc1EQ/EQ littermates obtained from Lamc1EQ/+ intercrosses. (D) Western analyses of a protein extract from a Lamc1EQ/EQ E7.5 whole embryo and Matrigel using an anti-laminin antibody under nonreducing conditions. (E, F) In situ binding of recombinant integrin α7x2β1 (green) in E7.5 WT or Lamc1EQ/+ (E) and Lamc1EQ/EQ (F) frozen sections. The RM was counterstained with an anti–laminin-α1 mAb (magenta). Bars, 100 μm. (G, H) Light microscopic images of control Lamc1EQ/+ (G) and Lamc1EQ/EQ (H) E7.5 whole embryos. The filled arrowhead indicates the PYS. The open arrowheads indicate trophoblast giant cells. Bars, 200 μm. (I–K) Immunofluorescence staining for cytokeratin-8 (magenta) and laminin (green) in WT or Lamc1EQ/+ (I) and Lamc1EQ/EQ (J, K) E7.5 sections. Blue, nuclei. The boxed area in J is magnified in K. The open diamonds in I indicate the blood sinus. The asterisks in I and J indicate trophoblast giant cells. The arrowheads in K indicate the extracellular matrix structure. Bars, 100 μm. Amn, amnion; Ch, chorion; ExE, extraembryonic ectoderm; Epi, epiblast; S, SexAI restriction site; P, PacI restriction site; TK, thymidine kinase gene; EPC, ectoplacental cone; EC, egg cylinder; PYS, parietal yolk sac.
- Figure S3. Secretion efficiency of recombinant laminin-111, laminin-511, and their EQ mutants.
SDS–PAGE under nonreducing conditions and subsequent immunoblot analyses of the conditioned media of human 293 cells transiently expressing laminin-111, laminin-511, or their EQ mutants. The anti–laminin-γ1 antibody detected secreted heterotrimeric laminins at around 800 kDa (arrowheads). Asterisks refer to the β1-γ1 dimer band. Conditioned medium of cells transfected with empty vectors (Mock) was also analyzed as a control.
- Figure 3. BM formation in Lamc1EQ/EQ zygotes.
(A–H) Whole-mount immunostaining of WT (A, C, E, G) and Lamc1EQ/EQ (B, D, F, H) blastocysts of E4.0 (A–D) and E4.3 (E–H) embryos. The asterisks indicate primitive endoderm cells. (I, J) Whole-mount immunostaining of E3.5 Lamc1EQ/+ (I) and Lamc1EQ/EQ (J) blastocysts cultured ex vivo for 48 h. Magenta, laminin; green, perlecan (A, B, E, F, I, J) or integrin α6 (C, D, G, H); blue, nuclei. Bars, 50 μm. The genotypes of the preimplantation embryos were determined by genomic PCR. (K, L) In situ binding of integrin α7x2β1 (green) to E5.5 embryonic sections. The RM was counterstained with an anti-laminin-α1 mAb (magenta). Blue, nuclei. (M, N) Immunofluorescence staining for laminin-α1 (cyan), Cdx2 (magenta), and Oct3 (green) in WT or Lamc1EQ/+ (M) and Lamc1EQ/EQ (N) E5.5 sections. The dashed lines indicate the region where extraembryonic ectoderm cells are associated with the BM. The open arrowheads indicate extraembryonic ectoderm cells dissociated from the BM. (O) Immunofluorescence staining for laminin (cyan) and laminin-α5 (green) in Lamc1EQ/EQ E5.5 sections. Blue, nuclei. The asterisks and open triangles indicate visceral endoderm cells and parietal endoderm cells, respectively. The arrowheads indicate the laminin-α5–positive epiblast BM. Bars, 50 μm. (P) Summary of the strategy for quantitative image analysis of RM length in the E5.5 egg cylinder. The RM length (double-headed arrow) was measured by image tracing. (Q) RM lengths measured in E5.5 Lamc1EQ/EQ embryos and control WT or Lamc1EQ/+ embryos. Data represent means ± SD. ***P < 0.001, significant difference by Welch's t test. The effect size was 3.13 and the statistical power was 1.0. ExE, extraembryonic ectoderm; Epi, epiblast.
- Figure 4. Generation of γ1 EQ conditional knock-in mice.
(A) Schematic representation of the generation of Lamc1cEQ. The open boxes represent exons. The protein coding sequences are indicated in gray. The targeting construct was designed to replace WT exon 28 encoding Glu at residue 1,605 with a floxed exon 28 followed by a mutated exon 28 encoding Gln at residue 1,605. The probes used for Southern blotting are indicated by bold lines. (B) Southern blot analyses of genomic DNA from WT, Lamc1cEQ(neo)/+, and Lamc1cEQ/+ offspring after digestion with SexAI and PacI. The detection of 9.0 and 7.4 kbp fragments with probe 1 and probe 2, respectively, in the Lamc1cEQ(neo)/+ lanes indicates occurrence of the expected homologous recombination. The detection of a 7.4 kbp fragment with probe 1 in the Lamc1cEQ/+ lane indicates that the neomycin-resistance gene has been removed from the Lamc1cEQ(neo) allele by the Cre-loxP system. (C) Genotypes of offspring obtained from Lamc1cEQ/+ intercrosses. (D–I) Histochemical analyses of Lamc1cEQ/cEQ (D–F) and Tie2-cre;Lamc1cEQ/cEQ (G–I) retinas. (D, G) In situ binding of recombinant integrin α3β1 (magenta) to frozen retinal sections. (E, H) Counterstaining of vascular BM with an anti–laminin-α5 antibody (green). Merged images with nuclear staining (blue) are also shown (F and I). Retinal vasculatures are indicated by filled (Lamc1cEQ/cEQ) and open (Tie2-cre;Lamc1cEQ/cEQ) arrowheads. Bars, 50 μm. (J) Genotypes of offspring obtained from mating between Lamc1cEQ/+ female and Tie2-cre;Lamc1cEQ/+ male mice. Only Lamc1cEQ/cEQ and Tie2-cre;Lamc1cEQ/cEQ mice are shown. S, SexAI restriction site; P, PacI restriction site; TK, thymidine kinase gene.
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