DEV02834 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Introduction of the Snail2 MO reduces the amount of Snail2 protein in the dorsal neural tube. Transverse sections through the trunk of a chick embryo with 21-23 somites at the axial level +4 and −1 somite, a region containing premigratory neural crest cells undergoing EMT. Embryos have been electroporated with either a 5-mismatch Snail2 control morpholino or Snail2 morpholino (MO; red) and stained for Snail2 protein (green). Upper panels show a representative section through three different embryos that have been transfected with the 5-mismatch Snail2 control MO; arrowheads identify a number of cells that are both red and green (yellow) and thus positive for the control MO and Snail2 protein. In embryos treated with the Snail2 MO, we detect very few red and green (yellow) cells and find the Snail2 MO to be, on average, 82% effective at prohibiting Snail2 translation. Moreover, the size of the Snail2 domain is reduced on the electroporated side, with Snail2 protein levels diminished by at least 33% compared with the contralateral control. Together, these data suggest that the Snail2 MO specifically inhibits translation of the Snail2 protein in developing neural crest cells of the avian embryo.

• Supplemental Figure 2 -

  Fig. S2. Depletion of Snail2 reveals changes in gene expression in premigratory avian neural crest cells at 15 hours post-electroporation. Responsiveness of various candidate genes to decreased Snail2 protein levels in the trunk after 15 hours of incubation with the Snail2 MO, as assessed by QPCR. Results are reported as fold difference relative to that obtained with the control MO. Black bars, Snail2 MO; white bars, control MO. Results presented are an average of at least two independent experiments performed with quadruple replicates for each condition (Snail2 or control MO).

• Supplemental Figure 3 -

  Fig. S3. Electrophoretic mobility shift and supershift assay identifying the specific interaction of Snail2 with E box 1 in the Cad6B regulatory region. The coding region of Snail2 was cloned in-frame into the expression vector pGEX-2T, and recombinant GST-Snail2 fusion protein, or GST protein as a control, was purified from BL21 bacterial lysate according to the manufacturer’s instructions (Pierce). Double-stranded ³³P-end-labeled E box 1-containing probes were incubated with or without affinity-purified recombinant GST-Snail2 protein in the presence or absence of various competitor DNA oligos. P, no protein; C, 500 ng GST protein. In the remaining lanes, 500 ng of affinity-purified recombinant GST-Snail2 protein was added to each binding reaction. S, no competitors added; WT and MUT, 10-fold and 100-fold molar excess of either unlabeled wild-type or unlabeled mutant E box 1 probes, respectively; CA, control antibody 50 ng of an irrelevant antibody (IgG); SA, Snail2 antibody 50 ng anti-Snail2 antibody (DSHB). Supershift reactions were pre-incubated for 15 minutes at room temperature prior to addition of the labeled probe. Retarded Snail2−E-box complexes and supershifted Snail2−E-box−antibody complexes are identified. Unbound probes are indicated by arrowheads.

• Supplemental Figure 4 -

  Fig. S4. Treatment with 10 μM of the proteasome inhibitor MG132 results in an increase in Snail2 protein in a stable cell line expressing Snail2. The chick Snail2 coding sequence was cloned into the pCMV-Tag2 mammalian expression vector (Stratagene) to produce an N-terminal Flag-tagged recombinant protein. To test the functional activity of the recombinant Flag-tagged Snail2 protein, the expression construct was injected into Xenopus embryos and, as expected, was found to induce excess neural crest cells as previously reported (LaBonne and Bronner-Fraser, 1998). Snail2-expressing stable cell lines were generated in the Human Embryonic Kidney 293T background (from the ATCC) followed by selection in 400 μg/ml G418 (geneticin; GIBCO-BRL). Single-cell dilutions were performed and clonal cell lines (20 in total) were allowed to grow for approximately 6 weeks in the presence of G418. (A) Three clonal cell lines were analyzed for Snail2 protein using an antibody to the Flag epitope (Sigma). Western blot analysis shows that two out of the three clonal lines produce Snail2 protein. (B) Clone 1 was subjected to treatment with the proteasome inhibitor MG132 (Sigma) or vehicle (100% methanol) in order to assess the half-life of the Snail2 protein. At various time points after treatment, cells were rinsed in 5 ml Ringer’s solution and harvested in 10 ml Ringer’s solution, followed by centrifugation at 1000 rpm for 5 minutes. 80 μg of total soluble protein was analyzed by immunoblotting to assess levels of Snail2 protein. The half-life of Snail2 was calculated to be approximately 19 minutes (the average of two independent MG132 experiments).