DEV013086 Supplementary Material

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• Supplemental Figure S1 -

  Fig. S1. Properties of Shh::GFP. (A) Western analysis of Shh proteins present in cell pellets and media fractions from cells transfected with Shh expression constructs. Cos-7 cells were transfected with pcDNA3 plasmid containing coding sequence for N-Shh, Shh or Shh::GFP, using Lipofectamine (Invitrogen). About 48 hours after transfection, the cells were rinsed with PBS twice and treated with 4 mM cyclodextrin in 0.5 ml OptiMEM (Invitrogen) at 37°C for 5 hours, the medium was collected and protein precipitated using trichloroacetic acid, with 2 mg BSA as a carrier. Cells were washed with PBS twice, and then lysed in TENT (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100) by scraping. Cell debris was removed by spinning the lysate for 25 minutes at 4°C and collecting only the supernatant. The protein samples from the medium and cell lysate were heated at 95°C for 10 minutes in SDS sample buffer (2% SDS, 10% glycerol, 0.1 M DTT, 60 mM Tris-HCl pH 8.0, 0.1% bromophenol blue) and used for standard SDS-PAGE in a 10% gel. The protein bands were blotted onto nitrocellulose membrane. The membrane was blocked in 5% skimmed milk in TBST (20 mM Tris-HCl pH 8.0, 500 mM NaCl, 0.05% Tween 20) for 3 hours. Primary antibody against the N-terminal domain of Shh Ab80 (Bumcrot et al., 1995) was applied at 1:200 dilution in TBST for 2 hours, washed in TBST four times for 5 minutes, and secondary antibody anti-rabbit horseradish peroxidase-linked whole antibody (donkey) was applied at 1:5000 for 1 hour. The membrane was washed and peroxidase reaction performed using ECL reagent (PerkinElmer). Western analysis of N-Shh (a truncated, non-cholesterol modified ligand), Shh and Shh::GFP proteins from cells confirms that Shh::GFP undergoes correct processing, although a comparison of the ratio of processed to un-processed Shh::GFP and Shh protein indicated that Shh::GFP processing was somewhat less efficient. To determine whether Shh::GFP ligand (N-Shh::GFPp) shows appropriate lipid-modification activities, we examined cell-surface retention in producing cells, a process that is cholesterol-dependent. As expected, western analysis of conditioned media demonstrated that only N-Shh was efficiently secreted into the medium. However, treatment of cells with cyclodextran, a ringed compound that extracts cholesterol from cells, resulted in efficient removal of both N-Shhp and N-Shh::GFPp from transfected cells into the media, whereas Shh and Shh::GFP precursor protein remained in the cell pellet. These biological properties indicate that N-Shh::GFPp was most likely cholesterol modified and cell-surface retained, comparable to its untagged, wild-type counterpart, N-Shhp. (B) N-Shh::GFPp activity was assayed in C3H10T1/2 cells, a mesenchymal stem cell line that expresses alkaline phosphatase (AP) in response to Shh signals (Kinto et al., 1997; Nakamura et al., 1997). Treatment of cells with increasing concentrations of N-Shhp or N-Shh::GFPp from conditioned media resulted in equivalent activation of AP activity indicating that these two forms shared similar specific activity. To address this more directly, we mutated the N-terminal cysteine where palmitoylation occurs (Pepinsky et al., 1998). This modification resulted in a sharp reduction of AP-inducing activity in both N-Shhp and N-Shh::GFPp at physiological concentrations (0-10 nM). As normal activity depends on N-terminal palmitoylation, these results indicate that N-Shh::GFPp was likely to be palmitoylated at its N-terminus, similar to wild-type Shh protein. (C) Western blots of fractions eluted from gel filtration chromatography probed with anti-Shh. Procedures for gel filtration were performed as described (Chen et al., 2004). Position of molecular-weight standards is shown; monomeric Shh proteins are indicated with green arrows and oligomeric forms with red. N-Shh::GFPp could form soluble multimeric complexes (SMCs), higher-order oligomeric complexes that have been suggested to mediate long-range Shh signaling activities and require both lipid modifications of Shh (Chen et al., 2004). Western analysis of gel filtration fractions reveals the presence of SMCs in both N-Shhp and N-Shh::GFPp conditioned media. However, N-Shh::GFPp continues to produce SMCs when the conserved amino acid for palmitoylation is mutated, suggesting that GFP may have some additional oligomerizing activity.

  Bumcrot, D. A., Takada, R. and McMahon, A. P. (1995). Proteolytic processing yields two secreted forms of sonic hedgehog. Mol. Cell. Biol.15, 2294-2303.

  Kinto, N., Iwamoto, M., Enomoto-Iwamoto, M., Noji, S., Ohuchi, H., Yoshioka, H., Kataoka, H., Wada, Y., Yuhao, G., Takahashi, H. E. et al. (1997). Fibroblasts expressing Sonic hedgehog induce osteoblast differentiation and ectopic bone formation. FEBS Lett.404, 319-323.

  Nakamura, T., Aikawa, T., Iwamoto-Enomoto, M., Iwamoto, M., Higuchi, Y., Pacifici, M., Kinto, N., Yamaguchi, A., Noji, S., Kurisu, K. et al. (1997). Induction of osteogenic differentiation by hedgehog proteins. Biochem. Biophys. Res. Commun.237, 465-469.

• Supplemental Figure S2 -

  Fig S2. GFP targeting of the Shh locus. (A) A targeted insertion was introduced into the murine Shh locus by homologous recombination in embryonic stem (ES) cells. Homologous integration of this vector into the Shh locus introduces GFP into the Shh protein immediately 3′ to the glycine-encoding triplet (position 198 in the amino acid sequence) and adds 10 amino acids downstream of GFP including the intein cleavage-cholesterol attachment site. A PGK-neo positive-selection cassette was flanked by FRT recognition sequences (gray triangles). This allele generates a GFP-tagged form of Shh protein (Shh::GFP). Exons 2 and 3 are shown as white boxes and the GFP coding sequence is shown as a green box. The 10 amino acid sequence containing the cholesterol attachment site (GC in red) is shown as a blue box. The location of PCR primers and probes that hybridize to particular restriction enzyme fragments used for genotyping are indicated. GFP, green fluorescent protein; PGK-Neo, phosphoglycerate kinase-neomycin positive-selection cassette; DTA, diphtheria toxin A negative-selection cassette. (B) The targeted Shh::gfp allele was identified in recombinant ES cells by Southern analysis of genomic DNA using probes that lie both upstream (5′ probe) and downstream (3′ probe) of targeting vector sequences. The 5′ probe hybridized to 6.1 kb and 8.4 kb NcoI restriction enzyme fragments generated from the Shh::gfp and wild-type alleles, respectively. The 3′ probe detected 10.5 kb and 11 kb SphI restriction fragments from the Shh::gfp and wild-type alleles, respectively. (C) Amplification products characteristic of the wild-type and Shh::gfp alleles were detected using a PCR assay. The primers (P1 and P2) located in intron 2 and exon 3. These primers amplify 250 and 1000 bp products from the wild-type and Shh::gfp alleles, respectively.

• Supplemental Figure S3 -

  Fig. S3. Activity and processing of Shh::GFP in vivo. (A-C) E10.5 embryos of indicated genotypes. (D-F) Skeletal preparations of E18.5 embryos. (G,H) Western analysis of Shh proteins from 293 cells and E9.5 embryos of indicated genotypes. Bands representing processed and unprocessed forms of Shh proteins indicated by arrows. Shh, full-length, unprocessed wild-type Shh protein; Shh::GFP, full-length unprocessed GFP-tagged Shh fusion protein; N-Shhp, N-terminal processed Shh active signaling moiety; N-Shh::GFPp, N-terminal processed form of GFP-tagged Shh protein (Shh::GFP ligand). Note the non-specific band just below the N-Shh::GFPp band in E9.5 embryos (asterisk).