Harney AS , Meade TJ , LaBonne C .

R01CA114058 NCI NIH HHS , U54CA151880 NCI NIH HHS , R01 CA114058 NCI NIH HHS , U54 CA151880 NCI NIH HHS

             epithelial to mesenchymal transition
             DNA binding transcription factor activity
             neural crest cell development

	Figure 3. Co(III)-Ebox inhibits Snail-mediated neural crest cell specification in Xenopus via a temperature-dependent mechanism. (A) In situ hybridization of Sox10 expression showing neural crest formation (left) and Twist expression showing neural crest migration (right) of embryos injected with Co(III)-Ebox into one cells of embryos at the 8-cell stage. Embryos were co-injected with β-galactosidase mRNA as a lineage tracer (red staining). Neural crest forming/migrating regions indicated with arrowheads on the injected side. (B, C) In situ hybridization showing the neural crest of control embryos or embryos injected with Co(III)-sb, Ebox, Co(III)-EboxMut or Co(III)-Ebox and grown at 14 (B) or 27 (C). Top panel: whole mount in situ hybridization showing FoxD3 expression in the neural crest and Krox20 expression in rhombomeres 3/5 of the CNS. For FoxD3 embryos reared at 14, Co(III)-sb n = 12; Ebox n = 16; Co(III)-EboxMut n = 16; and Co(III)-Ebox n = 13. For FoxD3 embryos reared at 27, Co(III)-sb = 13; Ebox n = 18; Co(III)-EboxMut n = 14; and Co(III)-Ebox n = 18. Bottom panel: expression of Sox10 in the neural crest and Krox20 (blue arrowheads). For Sox10 embryos reared at 14, Co(III)-sb n = 21; Ebox n = 33; Co(III)-EboxMut n = 35; and Co(III)-Ebox n = 38. For Sox10 embryos reared at 27, Co(III)-sb = 33; Ebox n = 35; Co(III)-EboxMut n = 35; and Co(III)-Ebox n = 36. Loss of Sox10 and FoxD3 is seen in the neural crest forming regions of embryos treated with Co(III)-Ebox grown at 27 (red arrowheads). (D) Cartoon representation of expected in situ hybridization result for Krox20 and neural crest markers (Sox10 and FoxD3) at stage 17. Black staining represents neural crest forming regions visualized by in situ hybridization for Sox10 or FoxD3 and indicated by red arrows. Pale blue staining indicates hindbrain forming regions of rhombomeres 3 and 5 visualized by in situ hybridization with Krox20 and indicated by blue arrows. (E) Cartoon representation of expected in situ hybridization result for Krox20 and neural crest markers (Sox10 and Twist) during neural crest cell migration. Black staining represents migrating neural crest regions visualized by in situ hybridization for Sox10 or Twist and indicated by red arrows. Pale blue staining indicates hindbrain forming regions of rhombomeres 3 and 5 visualized by in situ hybridization with Krox20 and indicated by blue arrows. (F) Co(III)-Ebox incubated in embryo lysate for 0 to 24 hours retains the and ability to inhibit XSnai2 DNA binding as visualized by EMSA.
	Figure 4. Co(III)-Ebox impairs neural crest cell migration in Xenopus embryos independently of neural crest specification. (A) In situ hybridization showing neural crest of embryos injected with Co(III)-Ebox or Co(III)-EboxMut during neurulation stages maintained at 14 (A, C) or 27 (B, D). (A, B) Co(III)-Ebox does not affect pre-migratory neural crest at 14 (A) or 27 (B) seen by in situ hybridization for FoxD3 (top panel) or Sox10 (bottom panel) For FoxD3 embryos maintained at 14, untreated n = 14; Co(III)-EboxMut n = 15; and Co(III)-Ebox n = 15. For embryos maintained at 27, untreated = 15; Co(III)-EboxMut n = 15; and Co(III)-Ebox n = 15. For Sox10 embryos maintained at 14, untreated n = 12; Co(III)-EboxMut n = 15; and Co(III)-Ebox n = 15. For embryos maintained at 27, untreated = 15; Co(III)-EboxMut n = 15; and Co(III)-Ebox n = 15. Krox20 is used to visualize non neural crest tissue in the hindbrain. (C) Embryos treated with Co(III)-Ebox have normal neural crest migration at 14. (D) Embryos treated with Co(III)-Ebox grown at 27 show impaired neural crest migration (red arrowheads) visualized by in situ hybridization for Twist and Krox20 (top panel) or Sox10 and Krox20 (bottom panel). For Twist embryos maintained at 14, untreated n = 14; Co(III)-EboxMut n = 14; and Co(III)-Ebox n = 14. For embryos maintained at 27, untreated n = 11; Co(III)-EboxMut n = 10; and Co(III)-Ebox n = 17. For Sox10 embryos maintained at 14, untreated n = 14; Co(III)-EboxMut n = 11; and Co(III)-Ebox n = 12. For embryos maintained at 27, untreated n = 13; Co(III)-EboxMut n = 12; and Co(III)-Ebox n = 14. (E, F) Quantification of normalized neural crest migration from Twist expression represented as means s.e.m. M = mandibular, H = hyoid, BA = anterior branchial, BP = posterior branchial. (E) T-tests determined no significant difference between embryos at 14. (F) T-tests determined that neural crest streams of embryos treated with Co(III)-Ebox are significantly different from controls [single star (*), M, P = 0.0002; H, P<0.0001; BA, P<0.0001 and BP, P<0.0001] and from embryos treated with Co(III)-EboxMut [double stars (**)] M, P = 0.0245; H, P = 0.0004, BA, P = 0.0005; and BP, P = 0.0001] at 27.
	Figure 5. Temperature modulation of Co(III)-Ebox-mediated Snail inhibition reveals a requirement for Snail in melanocyte formation. Neurula stage embryos injected with Co(III)-Ebox or Co(III)-EboxMut in the archenteron space were maintained at 14 and (A) neural crest migration visualized by whole mount in situ hybridization for Twist expression. Krox20 is used to visualize rhombomeres 3/5 in the hindbrain. Untreated n = 8; Co(III)-EboxMut n = 8; and Co(III)-Ebox n = 12. (B) Quantification of mandibular, hyoid and branchial streams of neural crest cells are represented as the distance migrated as a percentage of control embryos. T-tests determined that neither Co(III)-EboxMut nor Co(III)-Ebox had a significant effect on neural crest cell migration. (C) Sibling embryos were maintained until swimming tadpole stages at 14 (top panel) or 27 (bottom panel) when melanocyte formation could be assessed. Arrowheads point to regions of diminished melanocytes. For embryos maintained at 14, untreated n = 12; Co(III)-EboxMut n = 12; and Co(III)-Ebox n = 12. For embryos maintained at 27, untreated n = 12; Co(III)-EboxMut n = 12; and Co(III)-Ebox n = 13. (D) Same embryos were maintained until swimming tadpole stages at 14 (top panel) or 27 (bottom panel) when craniofacial cartilage formation could be assessed. Cartliage was stained with Alcian blue and dissected to visualize cartilage formation. (E) Injected embryos were reared at 14 (top panel) or 27 (bottom panel) until stage 28 and glial cell formation was visualized by whole mount in situ hybridization for FoxD3 expression. For embryos maintained at 14, untreated n = 15; Co(III)-EboxMut n = 17; and Co(III)-Ebox n = 11. For embryos maintained at 27, untreated n = 15; Co(III)-EboxMut n = 15; and Co(III)-Ebox n = 22. (F) Injected embryos were reared at 14 (top panel) or 27 (bottom panel) until stage 28 and primary neuron formation was visualized by whole mount in situ hybridization for N-tubulin expression. For embryos maintained at 14, untreated n = 15; Co(III)-EboxMut n = 17; and Co(III)-Ebox n = 13. For embryos maintained at 27, untreated n = 20; Co(III)-EboxMut n = 21; and Co(III)-Ebox n = 21.
	Figure 1. Co(III)-Ebox alleviates murine Snai1-mediated E-cadherin promoter repression.(A) Chemical structures; Co(III) Schiff base complex, Co(III)-sb; Ebox duplex oligonucleotide, Ebox; Co(III)-DNA conjugate with 2-base pair substitution in the Ebox region; Co(III)-EboxMut; Co(III)-DNA conjugate targeted to Snail factors, Co(III)-Ebox. (B) E-cadherin reporter gene activity in MCF7 cells expressing MSnail treated with Co(III)-Ebox from 0 to 15 nM. Data are represented as means ± s.e.m., n = 4. T-tests determined statistical significance from controls of 10 nM (P = 0.0034), 12.5 nM (P = 0.0060) and 15 nM (P = 0.0109) (*). (C) E-cadherin reporter gene activity in MCF7 cells expressing MSnail treated with 15 nM Co(III)-sb, Ebox, Co(III)-EboxMut or Co(III)-Ebox. Data are represented as means ± s.e.m., n = 4. T-tests determined statistical significance from controls of Co(III)-EboxMut (P = 0.0214) and Co(III)-Ebox (P = 0.0035) (*). Co(III)-Ebox to be significantly different from Co(III)-EboxMut (P = 0.0008) (**). (D) E-cadherin reporter gene activity in MCF7 cells transfected with pCS2+ (vector) treated with 15 nM Co(III)-sb, Ebox, Co(III)-EboxMut or Co(III)-Ebox. Data are represented as means ± s.e.m., n = 4. T-tests determined no statistical significance between the means. (E) MCF7 cell viability after treatment with Co(III)-Ebox at indicated concentrations after 24 h. Data are represented as means ± s.e.m., n = 3. T-tests determined no statistical significance between the means.
	Figure 2. Co(III)-Ebox binds target proteins to alleviate transcriptional repression in metastatic MDA-MB-231 cells.(A) E-cadherin reporter gene activity in MDA-MB-231 cells treated with Co(III)-Ebox from 0 to 40 nM. Data are represented as means ± s.e.m., n = 4. T-tests determined statistical significance from controls of 40 nM (P = 0.0489) (*). (B) E-cadherin reporter gene activity in MDA-MB-231 cells treated with 35 nM Co(III)-sb, Ebox, Co(III)-EboxMut or Co(III)-Ebox. Data are represented as means ± s.e.m., n = 4. T-tests determined statistical significance from controls of Co(III)-EboxMut (P = 0.0290) and Co(III)-Ebox (P = 0.0001) (*). Co(III)-Ebox to be significantly different from Co(III)-EboxMut (P = 0.0008) (**). (C) Luciferase reporter gene activity in MDA-MB-231 cells with EcadMut-luc reporter treated with 35 nM Co(III)-sb, Ebox, Co(III)-EboxMut or Co(III)-Ebox. Data are represented as means ± s.e.m., n = 4. T-tests determined no statistical significance between the means. (D) MDA-MB-231 cell viability after treatment with Co(III)-Ebox at indicated concentrations after 24 h. Data are represented as means ± s.e.m., n = 3. T-tests determined no statistical significance between the means. (E) Inhibition of Snail DNA binding in MDA-MB-231 cells after 24 h with 0 to 40 nM Co(III)-Ebox. A representative EMSA of triplicate samples is shown with Western blots of Snai1 and Actin. (F) A representative EMSA of MDA-MB-231 cells treated with shRNA targeting Snai1 with Western blots of Snai1 and Actin. (G) Quantification of the percentage of Slug probe bound to Snai1 in (E) by using a STORM 680 phosphoimager. Data are represented as the means ± s.e.m., n = 3.
	Figure 6. Proposed model for the temperature-dependent inactivation of proteins targeted by Co(III)-Ebox.

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