Western blot analysis on cellular proteins separated into nuclear and cytoplasmic fractions from sNF96.2 cells.

<p><b>A</b>: Whole cell lysate (W), cytosolic (C), and nuclear (N) fractions were immunoblotted with C-19 WT1 antibody. <b>B</b>: Results were expressed as optical density (O.D.). *p<0.05 relative to whole cell lysate.</p

siWT1 on cell proliferation.

<p><b>A</b>: Effects of 25 nM and 50 nM p-siWT1 and s-siWT1 on sNF96.2 cell proliferation at 48 and 72 hours. Data represented the average value of three independent transfection experiments. *p<0.05 compared to siNEG ones at same time. <b>B</b>: Growth curves of sNF96.2 cells treated with 25 nM and 50 nM siNEG, p-siWT1 or s-siWT1.</p

WT1 expression in sNF96.2 cells determined by immunocytochemistry.

<p>Immunofluorescence positive cells to 6F-H2 (<b>B</b>) and C-19 WT1 (<b>E</b>) antibody are merged (<b>C</b> and <b>F</b>, respectively) with their own DAPI stained nuclei (<b>A</b> and <b>D</b>). Scale bars: 50 µm.</p

siWT1 on apoptosis.

<p>Effects of siWT1 on cleaved caspase-3 expression in sNF96.2 cells treated with 50 nM for 48 and 72 hours determined by Western blot analysis. As a positive apoptosis control (PC), cell lysate of human fibroblasts exposed to 0.1 mM of staurosporine for 24 hours was used <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114333#pone.0114333-Johanssonn1" target="_blank">[83]</a>.</p

Time-course of siWT1 on sNF96.2.

<p>Western blot analysis on cells treated with 50 nM p-siWT1 (<b>A</b>) or s-siWT1 (<b>B</b>), compared to those treated with scrambled siNEG. siWT1 results were expressed as fold change compared to siNEG ones (<b>A1</b> and <b>B1</b> for p-siWT1 and s-siWT1, respectively). *p<0.05. <b>C</b>: Staining of WT1 protein in sNF96.2 cell treated with 50 nM siNEG, p-siWT1 or s-siWT1. Scale bars: 20 µm.</p

siWT1 on cell cycle.

<p><b>A</b>: Effects of siWT1 on PI3K/Akt/Cyclin D1 pathway in sNF96.2 cells treated with 50 nM p-siWT1 for 48 and 72 hours determined by Western blot analysis. <b>B</b>: Results were reported as fold change compared to siNEG ones (*p<0.05).</p

Additional file 3: of The phospholipase DDHD1 as a new target in colorectal cancer therapy

Table S1. Data from SWATH-MS Gene Ontology analysis. (XLSX 740 kb

Additional file 4: of The phospholipase DDHD1 as a new target in colorectal cancer therapy

Figure S2. Effects of DDHD1-expressing cells conditioned medium on DDHD1-silenced cell growth. Cell viability was measured by MTT assay on DDHD1-silenced SW480 cells in the presence of the conditioned medium (CM) of mock cells and DDHD1 overexpressing cells. (TIFF 3275 kb

Additional file 2: of The phospholipase DDHD1 as a new target in colorectal cancer therapy

Figure S1. DDHD1 silencing. To evaluate DDHD1 silencing a. Real-time PCR and b. Western blot analysis were performed on SW480, HCT116, HS5 and HUVEC transfected for 48 or 72 h with scrambled siRNA or DDHD1 siRNA. (TIFF 6629 kb

Immunophenotype of undifferentiated and differentiated cells.

<p>Immunofluorescence of undifferentiated GSCs neurosphere (left panels) and of differentiated cells (right panels) labelled with anti-human Sox2 (top panels), anti-human GFAP and anti-human TUBB3 (bottom panels).</p