Effects of CTO on cell adhesion molecules and cytokines mRNA expression.

<p>(I) CTO reverts the effects of CML exosome treatment on VCAM1, ICAM1 and IL8 mRNA expression in HUVEC cells. VCAM1 (a), ICAM1(b) and IL8 (c) mRNA expression increased in a dose dependent manner after adding exosomes (Exo) to endothelial cell monolayer. CTO (1-5-10 µM) reverts these effects in a time- and dose dependent manner. (II) VCAM1, ICAM1 and IL8 mRNA expression in HUVEC treated for 6 h either with low serum medium (CN), or with 50 µg/ml exosomes (Exo), or with 10 ng/ml of recombinant IL8 (Rec IL8) with or without CTO 10 µM, or with 50 µg/ml exosomes plus 10 µg/ml of a neutralizing anti-IL8 antibody (N Ab IL8). Values are representative for three independent experiments. *p≤0.05; **p≤0.01.</p

CTO inhibits the effects of LAMA84R exosomes on HUVEC migration.

<p>(a) Effects on migration of CTO-treated endothelial cells using 50 µg/ml of exosomes as chemoattractant. (b) 50 µg/ml of exosomes (Exo) with or without 10 µg/ml of neutralizing anti-IL8 antibody (N Ab IL8), or 10 ng/ml of recombinant IL8 (Rec IL8) with or without neutralizing anti-IL8 antibody were added as chemoattractants to the bottom wells., Motility of endothelial cells with or without increasing doses of CTO (1–10 µM) was evaluated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042310#s2" target="_blank">Material and Methods</a>. *p≤0.05; **p≤0.01.</p

<i>in vitro</i> inhibition of exosome-stimulated angiogenesis by CTO.

<p>(a) Phase contrast micrographs showing the effects of LAMA84R exosomes and CTO treatment on endothelial network formation (matrigel assay). Few cables are observed when HUVEC are plated in low serum medium (CN). Addition to HUVEC cells of 10 ng/ml of recombinant IL8 (Rec IL8) or 50 µg/ml of LAMA84R exosomes (Exo) induces the formation of capillary-like structures. No tube formation is observed when HUVEC are plated in the presence of 50 µg/ml of exosomes plus neutralizing anti-IL8 antibody (Exo + N Ab IL8). CTO inhibits the effects of recombinant IL8 (10 µM CTO + Rec IL8) or exosomes (Exo +10 µM CTO) on tube formation by HUVEC on matrigel. (b) Histograms showing the quantitative analysis of the cables length by Image J software.</p

Antitumor activity of CTO on human CML xenografts.

<p>LAMA84R cells were injected subcutaneously in NOD/SCID mice as described. After palpable tumor formation, mice were treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042310#s2" target="_blank">Material and Methods</a>. Comparison of the median tumor weight was used as index of the antitumor efficacy of the compounds.</p

CTO inhibits the adhesion of LAMA84R cells to exosome-treated HUVEC monolayer.

<p>(a) Phase contrast micrographs showing the adhesion of LAMA84R cells (arrows) on HUVEC monolayer treated with 50 µg/ml of exosomes (Exo) and with 50 µg/ml of exosomes after pre-treatment of 24 h with 10 µM CTO. (b) Adhesion of LAMA84R cells to endothelial cell monolayer treated for 6 h with: 50 µg/ml of exosomes (Exo) and exosomes plus increasing doses of CTO (1–10 µM); (c) Adhesion of LAMA84R cells to endothelial cell monolayer treated for 6 h with: 50 µg/ml of exosomes (Exo), 50 µg/ml of exosomes plus 10 µg/ml of a neutralizing anti-IL8 antibody (N Ab IL8), 10 ng/ml of recombinant IL8 (Rec IL8), 10 ng/ml of recombinant IL8 plus 10 µg/ml of a neutralizing anti-IL8 antibody and 10 ng/ml of recombinant IL8 with increasing doses of CTO (1–10 µM). Values are representative for three independent experiments. *p≤0.05; **p≤0.01.</p

LAMA84R exosomes characterization.

<p>(a) Exosomes released by LAMA84R cells observed with a scanning electron microscope. (b) Detection of Hsc70 and CD63 in 30 µg of cell lysate (lane 1) and 30 µg of exosomes lysate (lane 2). (c) Acetylcholinesterase assay. The activity of acetylcholinesterase, an exosome-specific protein marker, was determined in 10 µg either of total cell lysate (Cells) or of Exosomes (Exo); exosome-deprived conditioned medium (CM-Exo) and exosome-deprived Fbs (Fbs–Exo) were used as negative controls.</p

Effects of CTO on cell adhesion molecules and cytokines production.

<p>CTO inhibits the exosomes-stimulated increase of VCAM1, ICAM1 expression and IL8 secretion on HUVEC cells. Representative overlay histograms showing an increase of surface expression of VCAM1 (a) and ICAM1 (b) on HUVEC treated with 50 µg/ml of LAMA84R exosomes (blue line) compared to HUVEC treated with 50 µg/ml of LAMA84R exosomes plus 10 µM CTO (red line) or untreated HUVEC, as control (green line). (c) ELISA for IL8 release by HUVEC. Aliquots of conditioned medium (CM) from cells were collected after 6 h of stimulation either with 50 µg/ml of exosomes (CM HUVEC + Exo) or with 50 µg/ml of exosomes plus 10 µM CTO (CM HUVEC + CTO + Exo); low serum medium (CM HUVEC) or low serum medium plus 10 µM CTO (CM HUVEC + CTO) were used as negative controls. The amount of IL8 in 50 µg/ml of exosomes was also quantified.</p

CTO inhibits cell proliferation of LAMA84R and K562R cell lines.

<p>(a) Cell growth was measured by MTT assay after 24, 48, 72, 96 h of treatment with increasing doses of CTO (0.1-1-5-10 µM). The values were plotted as a percentage of the control (cells treated with DMSO). Each point represents the mean ± SD for three independent experiments. *p≤0.001. (b) CTO treatment decreases Bcr–Abl expression, inhibits Bcr–Abl tyrosine phosphorylation and its downstream substrate CrkL on LAMA84R and K562R cell lines. These cell lines were treated with increasing doses of CTO (0.1–5 µM) or DMSO (CN) for 72 h and 96 h; afterwards protein lysates were subjected to western blot analysis as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042310#s2" target="_blank">Material and Methods</a> using anti-phospho-Abl, anti-cAbl, anti-phospho-CrkL and anti-CrkL antibodies. Blots were then stripped and subsequently reprobed with antibody against β-actin to ensure equal loading.</p

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

CTO inhibits Akt and Erk 1/2 phosphorylation in exosomes-stimulated HUVEC.

<p>HUVEC were incubated with 50 µg/ml of exosomes with or without 10 µM CTO or with 10 ng/ml of IL8 with or without 10 µM CTO for 1 h (pErk 1/2 and Erk 1/2) or over night (pAkt and Akt). After the treatments, protein lysates were subjected to western blot analysis as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042310#s2" target="_blank">Material and Methods</a> using anti-phospho-Akt, anti- Akt, anti-phospho-Erk, anti-Erk antibodies. Blots were then stripped and subsequently reprobed with antibody against β-actin to ensure equal loading.</p