Absolute numbers of previously described and putative drivers identified in this study in different tumor types.

<p>Inset: color coding reflecting tumor types. Abbreviations: TNBC (Triple Negative Breast Cancer), HER2+BC (Her2-enriched Breast Cancers), ER+BC (ER-positive Breast Cancer), CRC (Colorectal Cancer), LUAD (Lung Adenocarcinoma), LUSC (Lung Squamous Cell Carcinoma), SCC (Squamous Cell Carcinoma), SRC (Soft Tissue Sarcoma), NET (Neuroendocrine Tumor), TC (Thymic Carcinoma), GC (Gastric Cancer), SIGRCA/KRUK (Signet Ring Adenocarcinoma/Krukenberg Cell Tumor), CUPO (Carcinoma of Unknown Primary Origin).</p

Left Panel: variants obtained from exome sequencing of tumor samples from Triple Negative Breast Cancer (TNBC) patients.

<p>Genes were grouped based on relevant biological activities/pathways. Right Panel: Variants identified as potential drivers (red: high-confidence drivers; gold: lower confidence drivers. Inset: Color coding scheme for types of mutations (mis-sense, termination, splice site, in-frame deletion), confidence of driver likelihood (high-confidence, lower confidence). Asterisks indicate mutations present in the COSMIC (<u>C</u>atalogue <u>o</u>f <u>S</u>omatic <u>M</u>utations in <u>C</u>ancer) database. The same coding scheme is used in Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194790#pone.0194790.g002" target="_blank">2</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194790#pone.0194790.g005" target="_blank">5</a>.</p

Novel putative drivers revealed by targeted exome sequencing of advanced solid tumors - Fig 2

<p><b>A</b>) Left Panel: variants obtained from exome sequencing of tumor samples from Her2-enriched Breast Cancer (HER2+ BRC) patients. Right Panel: Variants identified as potential drivers. <b>B)</b> Left Panel: variants obtained from exome sequencing of tumor samples from ER positive Breast Cancer (ER+ BRC) patients. Right Panel: Variants identified as potential drivers. Color codes are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194790#pone.0194790.g001" target="_blank">Fig 1</a>.</p

Previously described and putative drivers identified in this study grouped by functional classes or biological pathways.

<p>Pathway or functional class assignment was based on Gene Ontology supplemented by individual PathCards searches (<a href="http://pathcards.genecards.org/" target="_blank">http://pathcards.genecards.org/</a>) for each gene. Inset: color coding reflecting tumor types. Abbreviations: TNBC (Triple Negative Breast Cancer), HER2+BC (Her2-enriched Breast Cancers), ER+BC (ER-positive Breast Cancer), CRC (Colorectal Cancer), LUAD (Lung Adenocarcinoma), LUSC (Lung Squamous Cell Carcinoma), SCC (Squamous Cell Carcinoma), SRC (Soft Tissue Sarcoma), NET (Neuroendocrine Tumor), TC (Thymic Carcinoma), GC (Gastric Cancer), SIGRCA/KRUK (Signet Ring Adenocarcinoma/Krukenberg Cell Tumor), CUPO (Carcinoma of Unknown Primary Origin).</p

Left Panel: variants obtained from exome sequencing of tumor samples from Colorectal Cancer (CRC) patients.

<p>Right Panel: Variants identified as potential drivers. Color codes are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194790#pone.0194790.g001" target="_blank">Fig 1</a>.</p

Effect of 17β-estradiol treatment on Notch pathway components in HUVECs.

<p>(A) HUVECs were treated with 1 nM E2 or DMSO (V) for 24 hours under M4 experimental conditions (2% FBS overnight followed by 20% csFBS). Cell lysates were electrophoresed and immunoblotted with antibodies for Notch2, Notch4, Jagged1, Dll1. β-actin antibody was used to ensure equal loading. Densitometric analysis of Western blot assay is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071440#pone.0071440.s006" target="_blank">Figure S6B</a>. (B) HUVECs were treated with 1 nM E2 or DMSO (V) for 24 hours under M4 experimental conditions (2% FBS overnight followed by 20% csFBS). Total RNA was extracted and qRT-PCR analysis of Notch2, 4 and Jagged1 genes expression was performed. Relative changes in mRNA expression levels were calculated according to the 2^−ΔΔCt method using RPL13A as reference gene. Results are expressed as mean ± SEM of three independent experiments, each performed in triplicate. **P<0.01, significantly different from the control. (C) Western blot analysis for Notch4 in HUVECs after Notch4 (si)RNA treatment for 48 hours. Lysates were immunoblotted with Notch4 antibody. β-actin antibody was used to ensure equal loading. Densitometric analysis of Western blot assay is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071440#pone.0071440.s006" target="_blank">Figure S6C</a>. (D) qRT-PCR analyses were performed to detect reduction of Notch4 mRNA levels in HUVECs after Notch4 (si)RNA treatment for 48 hours. Relative changes in mRNA expression levels were calculated according to the 2^−ΔΔCt method using RPL13A as reference gene. Results are expressed as mean ± SEM of three independent experiments, each performed in triplicate. ***P<0.001, significantly different from the control.</p

E2 treatment counteracts Notch inhibition- induced vascular sprouting in collagen-based aortic ring explants.

<p>Aortic ring explants were embedded in collagen gels and cultured for 5 days in 2.5% csFBS medium containing 30 ng/ml of VEGF-A and treated with 1 nM E2, 5 µM DAPT or 1 nM E2 plus 5 µM DAPT. Treatment with DMSO (V) was used as control. Vascular sprouting was quantified by digital microscopy after 5 days of treatment by measuring the greatest length of sprouts from the body of the aortic ring at three distinct points per ring and on three rings per treatment. One representative picture of three different experiments is shown (A) with the respective sprout lengths (B). Data are expressed as mean ± SEM. **P<0.01, DAPT significantly different from the control, §P<0.05, E2 plus DAPT significantly different from DAPT.</p

E2 treatment counteracts Notch inhibition- induced endothelial branching <i>in vitro</i>.

<p>HUVECs were hormone-stripped and treated overnight with 1 nM E2, 5 µM DAPT and a combination of the two. Treatment with DMSO (V) was used as control. The following day 9×10⁴ cells were seeded on 400 µl of Matrigel and treatment continued for additional 8 hours. Number of closed circles was quantified in eight fields after 8 hours of network formation. One representative picture of three different experiments is shown (A) with the respective counts (B). Data are expressed as mean ± SEM. **P<0.01, DAPT significantly different from the control, §P<0.05 E2 plus DAPT significantly different from DAPT.</p

Effect of 17β-estradiol treatment on Notch1 processing in HUVECs under different experimental conditions.

<p>(A) HUVECs were treated with 1 nM 17β-estradiol (E2) or DMSO (V) for 24 hours under different experimental conditions as described in the Materials and Methods section. Cell lysates were electrophoresed and immunoblotted with Notch1 (C-20) antibody to detect changes in the transmembrane form of Notch1 (Notch1TM). β-actin antibody was used to ensure equal loading. (B) Densitometric analysis of Western blot assay to quantify Notch1TM protein levels. Results are expressed as mean ± SEM of three independent experiments, **P<0.01, ***P<0.001 significantly different from the control. (C) HUVECs at different confluence (70% vs. 90%) were treated with 1 nM E2 or DMSO (V) for 24 hours under M4 experimental conditions (2% FBS overnight followed by 20% csFBS). Cell lysates were electrophoresed and immunoblotted with Notch1 (C-20) antibody to detect changes in the precursor (Notch1 precursor) and the transmembrane (Notch1TM) form. β-actin antibody was used to ensure equal loading. Densitometric analysis of Western blot assay is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071440#pone.0071440.s006" target="_blank">Figure S6A</a>. (D) HUVECs were treated with 1 nM E2 or DMSO (V) for 3, 6 and 24 hours under M4 experimental conditions (2% FBS overnight followed by 20% csFBS). Total RNA was extracted and qRT-PCR analysis of Notch1 gene expression was performed. Relative changes in mRNA expression levels were calculated according to the 2^−ΔΔCt method using RPL13A as reference gene. Results are expressed as mean ± SEM of three independent experiments, each performed in triplicate.</p

Proposed model for E2-mediated activation of VEGF-A-Dll4-Notch1 signalling and consequences on sprouting angiogenesis in HUVECs.

<p>According to a widely accepted model <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071440#pone.0071440-Sainson1" target="_blank">[5]</a> Vascular Endothelial Growth Factor A (VEGF-A), by binding to VEGF Receptor-2 (VEGF-R2), promotes sprouting on endothelial cells and induces expression of Dll4 which, by activating Notch1 in adjacent cells leads to reduction of VEGF-R2 and inhibition of sprouting (A). E2 treatment enhances VEGF-A-mediated increase of the active form of Notch1 (N1IC), effect antagonized by the estrogen receptor antagonist ICI 182.780. In the presence of active Notch1 signalling, a further increase of N1IC induced by E2 does not affect sprouting (B). Notch1 inhibition by treatment with DAPT causes increased sprouting (C). Co-treatment with E2 counteracts DAPT-induced increase in vascular sprouting through still undetermined mechanisms (D).</p