15 research outputs found

    Selection of the clones used to validate the screen strategy.

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    <p>Selection of the clones used to validate the screen strategy.</p

    Whole-mount <i>in situ</i> hybridisation images on clones with regionalised expression patterns.

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    <p>For each clone the corresponding clone number and <i>Xenopus</i> gene symbol are shown. Vegetal view (stage 10.5 except for <i>foxh1</i>, which is side view); dorsal view (stage 15 and 20, posterior is up); lateral view (stage 30, anterior is to the left).</p

    Proof of principle of the screen.

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    <p>(A) 12 pools, each one with one clone of known activity, were selected from the full-length EST library and injected into embryos as described. Protein extracts from collected embryos were subjected to Western blot using indicated antibodies to observe phosphorylation changes of specific signalling molecules at blastula and gastrula stages. Note the reduction of phospho-Smad2 activity at gastrula stage on pool 2, and reduction of phospho-Smad1/5/8 activity on pool 11. (B) De-convolution of pool 2. <i>cerberus</i> is identified as a negative regulator of Smad2 (pSmad2, lower panel) but not of Smad1 (pSmad1, upper panel) phosphorylation at gastrula stage. (C) De-convolution of pool 11. <i>wnt8a</i> is identified as a negative regulator of Smad1/5/8 phosphorylation (pSmad1, middle panel) and activator of Wnt signalling (pLRP6, upper panel) at gastrula stage. UI: uninjected.</p

    Classification of the positive clones identified in the screen.

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    <p>Values were given as percentages against total clone numbers identified in the screen (<i>n</i> = 20).</p>a<p>Percentage of clones having at least one publication describing their functions in <i>Xenopus</i>.</p>b<p>6 functional groups were established as described.</p>c<p>A total of 20 positive clones have been identified. Clones that modulate the activities of more than one signalling pathways, are counted in each group.</p

    Flowchart of the experimental procedure of the screen.

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    <p>A <i>X. tropicalis</i> library of unique, full-length clones has been established based on sequence comparison and clustering of over 1,220,000 ESTs, and rearrayed in a 96-well plate format. Pools of 8 mRNAs were prepared from pooled bacteria culture and <i>in vitro</i> transcription. Then <i>in vitro</i> transcribed mRNA pools were injected into fertilized <i>X. laevis</i> embryos at 1–2 cell stage. After microinjection, injected embryos were collected at stage 8 (blastula), stage 10.5 (gastrula), and stage 14 (neurula). Protein extracts from embryos were loaded onto SDS-PAGE for subsequent Western blot analysis. Antibodies used include anti-phospho-Smad1/5/8, anti-phospho-Smad2, anti-phospho-Akt, and anti-phospho-Erk. Once a potential active pool was identified, the pool was de-convoluted and single molecule injection was performed to identify the active molecule.</p

    Kinetics of the activation of signalling molecules during early <i>Xenopus</i> development.

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    <p><i>X. laevis</i> embryos were collected at the time indicated and subjected to Western blot analysis. Membranes were probed with anti-phospho-Smad1/5/8 (pSmad1) antibody for monitoring BMP activity, anti-phospho-Smad2 (pSmad2) antibody for TGF-β/Nodal signalling, anti-phospho-Erk (pErk) for MAPK/Erk signalling and anti-phospho-Akt (pAkt) for PI3K/Akt signalling. Anti- Smad2, anti- Akt, and anti-Erk were used as loading controls to ensure all lanes have been loaded equally.</p

    Summary of the positive clones identified in the screen.

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    <p>Clones that have not been previously reported in the literature for their roles in regulating signalling events are shown in <b>bold</b>.</p>a<p>Notes describes additional effects on the activities of different signalling pathways.</p

    Characterisation of phospho-specific antibodies.

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    <p>(A) Characterisation of anti-phospho-Akt (pAkt) and anti-phospho-Erk (pErk) antibodies, the PI3K/Akt inhibitor LY294002 and FGF inhibitor SU5402 were used to inhibit Akt and Erk phosphorylation in gastrula embryos, respectively. 1% DMSO was used to exclude any possible interference from the inhibitor solvent. Anti-Erk (Erk), anti-Akt (Akt) and anti-α-tubulin (α-tubulin) were used as loading controls. (B) Characterisation of anti-phospho-Smad1/5/8 (pSmad1) and anti-phospho-Smad2 (pSmad2) antibodies. The TGF-βRI inhibitor SB505124 was used to inhibit Smad2 phosphorylation in gastrula embryos; injection of <i>wnt8a</i> mRNA was used to inhibit <i>bmp4</i> expression, thus preventing Smad1/5/8 phosphorylation. All inhibitors have been added at stage 6. 1% DMSO was used to exclude any possible interference from the inhibitor solvent. Smad2 and α-tubulin serves as internal controls to ensure equal loading in all lanes.</p

    Examples on identification and de-convolution of active regulators.

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    <p>(A–B) identification of the MAPK/Erk activator <i>fbxo43</i> (<i>erp1</i>). (A) Western blot of stage 8 embryos injected with 12 pools (01–12) derived from plate #01 and 7 pools (01–07) from plate #02 and probed with anti-phospho-Erk (pErk) antibody. The arrow indicates increased Erk phosphorylation upon injection of mRNAs derived from plate #02, pool 07. (B) De-convolution of the above pool. Embryos injected with single RNAs were collected at stage 8 and uninjected lysate from stage 8 and stage 10.5 were used as negative and positive control of Erk phosphorylation respectively. The arrow indicates the active clone of TEgg009F05, identified in plate #2, column 08, row G. This clone was confirmed as the <i>X. tropicalis fbxo43</i> (<i>erp1</i>) gene. (C–D) Identification of PI3K/Akt inhibitor <i>prkaca</i>. (C) Western blot of stage 10.5 embryos injected with 24 pools (01–12) derived from plate #09 and #10 and probed with anti-phospho-Akt (pAkt) antibody. The arrow indicates decreased Akt phosphorylation upon injection of mRNAs derived from plate #10, pool 02. (D) De-convolution of the above pool. mRNA synthesis, injection, and Western blot were performed as in (B) except that stage 10.5 embryos were used. The arrow indicates the active clone of TEgg046d13 is identified in plate #09, column 02, row E. This clone was later identified as encoding the <i>X. tropicalis prkaca</i> gene.</p

    TRH stimulates re-epithelialisation and enhances proliferation in wounded human skin.

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    <p>(<b>A</b>) Representative H&E stained sections of human skin punch-wounds following 6 days culture with either vehicle control or 10 ng/mL TRH. New epithelial tongues indicated by the white-hatched lines. Scale bars represent 50 µm. (<b>B</b>) Representative images of Ki-67-TUNEL double-stained sections of human skin punch wounds following 6 days culture with either vehicle control or 10 ng/mL TRH. The white-hatched line demarcates new epithelial tongue. (<b>C</b>) The graph displays length measurements of the new epithelial tongue (as demarcated by the white-hatched lines in <b>A</b>) as analysed from H&E stained human skin sections following treatment with vehicle control, 5 ng/mL and 10 ng/mL TRH. (<b>D</b>) Percentage of proliferative (Ki67-positive) cells in the new epithelial tongue of vehicle control, 5 ng/mL and 10 ng/mL TRH-treated human skin wounds. (<b>E</b>) Percentage of apoptotic (TUNEL-positive) cells in the new epithelial tongue of vehicle control, 5 ng/mL and 10 ng/mL TRH-treated human skin wounds. Data are mean ± SEM of 4 female donors. Significance relative to control data at the same time-point denoted by *P<0.05, **P<0.01, ***P<0.001.</p
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