Verification of the improved method in E8.5 <i>miR-451</i>-<i>LacZ</i> embryos.

<p>The E8.5 <i>miR-451</i> promoter-<i>LacZ</i> embryos were generated from mating between transgenic male <i>miR-451</i> promoter-<i>LacZ</i> mice and wildtype female SW mice. (<b>A</b>) Wildtype and <i>LacZ</i> embryos stained with the X-gal/FeCN method. (<b>B</b>) Wildtype and <i>LacZ</i> embryos stained with the S-gal/TNBT method. (<b>C</b>) Wildtype and <i>LacZ</i> embryos stained with the improved method. The blood islands are indicated with arrowheads. Additional positive signals at notochord and endodermal lining of prospective midgut region are indicated with an arrow.</p

Verification of the improved method in E13.5 embryonic tissue sections.

<p>(<b>A</b>) Section stained with the X-gal/FeCN method. (<b>B</b>) Section stained with the S-gal/TNBT method. (<b>C</b>) Section stained with the improved method.</p

Verification of the improved method in E10.5 <i>miR-322/503</i>-<i>LacZ</i> embryos.

<p>(<b>A</b>) Wildtype and <i>LacZ</i> embryos stained with the X-gal/FeCN method. (<b>B</b>) Wildtype and <i>LacZ</i> embryos stained with the S-gal/TNBT method. (<b>C</b>) Wildtype and <i>LacZ</i> embryos stained with the improved method. The heart and somites are indicated with arrowheads. The signals around the cranial mesoderm and arches are indicated with an arrow.</p

Optimization of β-galactosidase staining.

<p>Optimization of β-galactosidase staining.</p

Comparing the X-gal/FeCN and S-gal/TNBT methods in staining <i>miR-322/503</i>-<i>LacZ</i> positive E8.5 embryos.

<p>(<b>A</b>) Wildtype and <i>LacZ</i> embryos stained with the X-gal/FeCN method overnight. (<b>B</b>) Wildtype and <i>LacZ</i> embryos stained with the S-gal/TNBT for 20 minutes. (<b>C</b>) Wildtype and <i>LacZ</i> embryos stained with the X-gal/FeCN method for two weeks. (<b>D</b>) Wildtype and <i>LacZ</i> embryos stained the S-gal/TNBT method for two weeks. The cardiac bulge is indicated with arrowheads.</p

Schematic diagram of the <i>miR-322/503</i> knockout locus and the mating schedule.

<p>(<b>A</b>) <i>Mirc24</i>^{<i>tm1Mtm</i>}/ Mmjax mice were crossed with Tg(<i>Sox2-Cre</i>) mice. Due to the presence of three LoxP sites, there were three possible alleles resulted from Cre-mediated recombination. We selected the one (dotted-box) with the <i>miR-322/503</i> stemloop ablated to carry out further mating and named it “<i>miR-322/503</i> KO”. (<b>B</b>) E8.5 embryos were produced from mating of male hemizygous knockout of <i>miR-322/503</i> (<i>miR-322/503</i>^-/Y) with female wildtype SW mice. Half of the embryos are expected to be heterozygous knockout female mice (<i>miR-322/503</i>^-/+) and also <i>LacZ</i> positive.</p

Structural diversity analysis of 7365 non-flat fragment-sized natural products based on pharmacophore (orange) and radial (red) fingerprints.

<p>Using SOMs (generated by training non-flat structures subset) we analyzed the distribution and therefore the coverage of the total diversity of non-flat fragments by the subsets clustered on the number of rings present in the structure.</p

Distribution of compounds within the SOMs trained using ECPF_4 fingerprints of 20185 fragment-sized natural products (Fig 3A and 3B) and 7365 non-flat fragment-sized natural products (Fig 3C–3H).

<p><b>(a)</b> 20185 fragment-sized natural products. <b>(b)</b> 7365 non-flat fragment sized natural products (F<i>sp</i>³* > 0.45). <b>(c)</b> 7365 non-flat fragment-sized natural products. <b>(d)</b> 1-ring molecules; 37% coverage of non-flat fragments. <b>(e)</b> 2-ring molecules; 68% coverage of non-flat fragments. <b>(f)</b> 3-ring molecules; 56% coverage of non-flat fragments. <b>(g)</b> 4-ring molecules; 21% coverage of non-flat fragments. <b>(h)</b> 5-ring molecules; 2% coverage of non-flat fragments. Each cell represents a cluster of fragments and the distance between cells (i.e. nearby cell are structurally related compounds) is indicated by the shading of the cell borders; darker borders indicate larger distance. Cells are coloured by population, with white for empty cells, and red for cell containing more than 5 compounds. The trained SOM is characterized by a toroidal architecture, which means that the top edge is connected to the lower edge and the left edge with the right edge.</p

Pharmacophore analysis of natural products.

<p>Based on the number of unique pharmacophore triplets (1–6 bonds), generated using eight features, we identified that fragment-sized natural products cover ~66% of the unique pharmacophore of the whole DNP.</p

Distribution of 2-ring fragments dataset within the non-flat fragment-sized natural products SOM.

<p>For a few selected cells are, the most representative structures (green circle) is shown. In the black square box is reported one example of the molecular similarity within the same cell. The entire list of seed compounds can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120942#pone.0120942.s003" target="_blank">S3 Table</a>.</p