DEV027805 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Conditional knockout scheme and genotyping. (A) Schematic of the conditional knockout scheme and the genotyping strategy. The two loxP sites are inserted around exon 3 of the α3 integrin gene. In addition, there is an Frt site between exons 2 and 3, which was left over after deleting the selective neo cassette. (B) Genotyping PCR reactions. In order to distinguish the targeted from the wild-type allele, primers in exons 2 and 3 were used. For the WT allele, this generates a 542 bp product, and for the conditional KO (CKO) allele a 623 bp product; in tissues where Cre has been expressed, there is no PCR product. Deleting exon 3 creates three termination codons in exon 4. Details of the PCR primers and genotyping protocol are available on request. Sample PCR reactions are shown. Top panel, α3 integrin-null allele, where the top band indicates a null allele and the bottom band a wild-type or CKO allele. Upper middle panel, PCR for Cre allele. Lower middle panel, PCR reaction of CKO allele, showing one CKO allele and one wild-type allele. Bottom panel, CKO allele PCR, showing the difference between a CKO/CKO and wild-type genotype.

• Supplemental Figure S2 -

  Fig. S2. Tcf-luciferase reporter (TOPFLASH) assays in cell lines. (A) WT, wild-type immortalized collecting duct cells; KO, α3 integrin mutant cells; LN-mut, KO cells stably expressing an α3 integrin subunit with a mutation in the laminin-binding domain (previously published as G165A) (Zhang et al., 2003); the α3-stalk and α6-stalk constructs were described previously and are chimeric cDNAs for integrin α subunits expressing portions of the α3 and α6 subunits (Yauch et al., 2000). α3-stalk expresses the α3 extracellular domain and α6 cytoplasmic domain, whereas α6-stalk expresses the α3 extracellular domain until the beginning of the stalk (juxtamembrane) domain, and then switches to the α6 stalk and cytoplasmic domains. All luciferase results are normalized by co-transfection with a β-galactosidase expression construct under control of a constitutively active promoter. (B) Tcf-luciferase (TOPFLASH) assays with the WT (B7) cell line, showing Tcf-luciferase activity is sensitive to Wnt blockade with Fz8CRD, Dkk1, or transfection with an Axin-expressing plasmid that leads to destabilization of β-catenin.

  Reference

  Yauch, R. L., Kazarov, A. R., Desai, B., Lee, R. T. and Hemler, M. E. (2000). Direct extracellular contact between integrin alpha(3)beta(1) and TM4SF protein CD151. J. Biol. Chem. 275, 9230-9238.

• Supplemental Figure S3 -

  Fig. S3. Tcf-Luciferase reporter (TOPFLASH) assays in cell lines. (A,B) Cells are as described for Fig. S2. WTCM, wild-type conditioned medium, that is medium removed from wild-type cells after 24 hours of culture. WTCM rescues luciferase activity in KO cells and the rescue is sensitive to Wnt blockade with Fz8CRD, Dkk1, or transfection with an Axin-expressing plasmid.

• Supplemental Figure S4 -

  Fig. S4. Effect of Wnt and Hgf on cell survival in kidney papillae. Low-magnification images are shown. Isolated papillae were sectioned and TUNEL stained to reveal apoptotic cells prior to (A) or after (B,C) culture under various conditions. WT, wild-type papillae; KO, α3β1 integrin-deficient papillae. TUNEL stain, green. The genotype is designated in the upper left corner of each panel, the treatment in the upper right corner. (A) Papillae directly sectioned without organ culture. Significantly more apoptosis was observed in α3β1 KO than in WT kidney papillae. (B) Effect of Wnt3a-conditioned medium and Wnt blockade. Wnt3a prevented apoptosis in KO papillae. Wnt blockers Dkk1 and Fz8CRD stimulated apoptosis in WT papillae; the control is conditioned medium from a vector expressing only the Fc region used in the Fz8CRD construct. (C) The effect of Hgf- or IGF-neutralizing antibodies or Hgf on cell survival. The Hgf-neutralizing antibody stimulated apoptosis in WT papillae. Hgf did not prevent apoptosis in KO papillae. These images are from a separate experiment to that shown in Fig. 7.

• Supplemental Figure S5 -

  Fig. S5. Cell proliferation in WT and KO kidneys. Proliferation was assessed by Ki67 staining. (A,B) Low-magnification (4×) images of WT (A) and KO (B) kidneys. The overall appearance of proliferation was similar in WT and KO kidneys, with the densest proliferation in the peripheral nephrogenic zone. (C,D) Higher-magnification images taken from the base of the papilla of WT (C) and KO (D) show that there was little proliferation at the very tip of the papilla in WT kidneys. In the mid-section of the WT papilla and its equivalent area in KO kidneys, Ki67-positive cells were present in collecting ducts (CD) at similar frequency in WT and KO kidneys (arrows). Ki67-positive cells were also abundant in loops of Henle (LH) that descend from the cortex into the papilla.