Supplementary Materials for

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1 Supplementary Materials for Phosphoproteomic Analysis Implicates the mtorc2-foxo1 Axis in VEGF Signaling and Feedback Activation of Receptor Tyrosine Kinases Guanglei Zhuang, Kebing Yu, Zhaoshi Jiang, Alicia Chung, Jenny Yao, Connie Ha, Karen Toy, Robert Soriano, Benjamin Haley, Elizabeth Blackwood, Deepak Sampath, Carlos Bais, Jennie R. Lill, Napoleone Ferrara* *Corresponding author. Published 16 April 2013, Sci. Signal. 6, ra25 (2013) DOI: /scisignal This PDF file includes: Fig. S1. KinomeView profiling of endothelial cells. Fig. S2. Quality control of phosphoproteomic experiments. Fig. S3. Regulation of PI3K-mTORC2-Akt-FoxO1 axis by VEGF. Fig. S4. Different classes of small-molecule inhibitors used in the study. Fig. S5. VEGF signaling mechanisms to regulate phosphorylation. Fig. S6. mtor kinase inhibitors in endothelial RTK reprogramming. Fig. S7. Feedback activation of endothelial RTKs. Fig. S8. PI3K inhibitors in endothelial RTK reprogramming. Other Supplementary Material for this manuscript includes the following: (available at Table S1 (Microsoft Excel format). The complete data set for identified phosphopeptides. Table S2 (Microsoft Excel format). VEGF-regulated phosphopeptides and membership values in fuzzy c-means clustering. Table S3 (Microsoft Excel format). Gene ontology and pathway analysis of the VEGF-regulated proteins. Table S4 (Microsoft Excel format). Gene list and sirna sequences for RNAi screen. Table S5 (Microsoft Excel format). Kinase enrichment analysis of the VEGFregulated phosphoproteins. Table S6 (Microsoft Excel format). Microarray analysis of HUVECs stimulated with VEGF. Table S7 (Microsoft Excel format). NanoString ncounter analysis of HUVECs stimulated with VEGF.

2 Table S8 (Microsoft Excel format). Microarray analysis of HUVECs treated with mtor kinase inhibitors.

3 Figure S1. KinomeView profiling of endothelial cells. A. Experimental overview. Illustration reproduced courtesy of Cell Signaling Technology, Inc. ( B. HUVECs were treated with VEGF (50 ng/ml) or sorafenib (50 µg/ml) and then analyzed by Western blot with the indicated phospho-motif antibodies. Data represent two independent experiments.

4 Figure S2. Quality control of phosphoproteomic experiments. A. Scatter plots of raw data showed linear correlation between control and VEGF-treated HUVECs. B. Distribution of changes in phosphorylation from control or sorafenib-treated HUVECs. Scatter plot of raw data is inset. Phospho-peptides with decreased (zscore < 1.5) or increased (z-score > 1.5) phosphorylation are indicated. C. Distribution of changes in phosphorylation from control or VEGF-treated HUVECs

5 across different time points. Phospho-peptides with decreased (z-score < 1.5) or increased (z-score > 1.5) phosphorylation are indicated.

6 Figure S3. Regulation of PI3K-mTORC2-Akt-FoxO1 axis by VEGF. A. Quantification of VEGF-induced would closure (n=3 biological replicates; *p<0.01, Kruskal-Wallis test followed by Mann-Whitney tests). B. Mass spectra of a phospho-peptide from FoxO1. C. HUVECs were stimulated with VEGF (50 ng/ml) as indicated. Components of the PI3K-mTORC2-Akt pathway were immunoblotted with phospho-specific antibodies. Data represent two independent experiments.

7 Figure S4. Different classes of small-molecule inhibitors used in the study. The concentrations were determined for the inhibitors to inhibit corresponding pathways upon VEGF activation. Data represent two independent experiments.

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9 Figure S5. VEGF signaling mechanisms to regulate phosphorylation. A. HUVECs were pre-treated with PI3K inhibitors (0.5 µm), stimulated with VEGF (50 ng/ml), and analyzed by Western blot with the AKT motif antibody. Data represent two independent experiments. B. HUVEC viability in the presence of emerolimus was measured by CellTiter-Glo luminescent assays (n=8 biological replicates). C. Raptor, rictor or mtor was knocked down in HUVECs and cell viability was assessed by CellTiter-Glo luminescent assays (n=8 biological replicates). D. Quantification of HUVEC sprouts in response to different inhibitors (n=3 biological replicates; *p<0.01, ANOVA followed by Dunnett tests). E. Microarray analysis of the VEGF gene signature (n=3 biological replicates). F. The ratio of the amount of FoxO1 Thr 24 to that of total FoxO1 (n=3 biological replicates; *p<0.05, Kruskal-Wallis test followed by Mann-Whitney tests). G. Percentage of HUVECs that showed FoxO1 nuclear localization (n=4 biological replicates; *p<0.01, Kruskal-Wallis test followed by Mann-Whitney tests).

10 Figure S6. mtor kinase inhibitors in endothelial RTK reprogramming. A. Protein phosphorylation following PI3K inhibition was probed with AKT motif antibody. Data represent two independent experiments. B. Western blot analysis of total protein abundance. Data represent two independent experiments. C. The ratio of the amount of phospho-proteins to that of total proteins (n=3 biological replicates).

11 Figure S7. Feedback activation of endothelial RTKs. A. The ratio of the amount of phospho-proteins to that of total proteins; and the ratio of the amount of total proteins to that of actin (n=3 biological replicates). B. Gene expression of RTKs was analyzed by quantitative PCR (n=4 biological replicates).

12 Figure S8. PI3K inhibitors in endothelial RTK reprogramming. A. The ratio of the amount of phospho-proteins to that of total proteins; and the ratio of the amount of total proteins to that of Actin (n=3 biological replicates). B. HUVECs were treated with GDC-0980, GDC-0941 or everolimus (0.5 µm) for 24 hours, and cell lysates were analyzed by phospho-rtk antibody array. Data represent two independent experiments. C. HUVECs were treated with GDC-0941 (0.5 µm). Cell lysates were analyzed by immunoblotting with indicated antibodies. Data represent two independent experiments.