Supplementary Data

Title: mTORC2 MEDIATES CXCL12-INDUCED ANGIOGENESIS

Authors: Mary E. Ziegler1, Michaela M.S. Hatch1, Nan Wu1, Steven A. Muawad1 and Christopher C.W. Hughes1,2,3,4

Affiliations:1The Department of Molecular Biology and Biochemistry, University of California Irvine, Irvine, CA 92697, USA2The Department of Biomedical Engineering, University of California Irvine, Irvine, CA 92697, USA3The Edwards Lifesciences Center for Advanced Cardiovascular Technology, University of California Irvine, Irvine, CA 92697, USA4Corresponding Author: Christopher C.W. Hughes Ph.D. cchughes@uci.edu

Journal: Angiogenesis

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Fig. S1 Optimization of CXCL12 stimulation

(a) Serum-starved EC were stimulated with CXCL12 (50 ng/ml) at various time points

(as indicated). Cell lysates were harvested and a Western blot was performed to

determine the phosphorylation of Akt (S473) and S6RP (S240/244). -actin was used as

a loading control. (b) Serum-starved EC were stimulated with CXCL12 for 30 min using

a range of concentrations (as indicated). Cell lysates were harvested and a Western blot

was performed to determine the phosphorylation of Akt (S473) and S6RP (S240/244). -

actin was used as a loading control. The experiment was repeated a minimum of 3 times

with similar results.

2

Fig. S2 CXCL12 does not significantly activate mTORC1

Serum-starved EC were either left untreated or stimulated with CXCL12 (50 ng/ml, 30

min). The lysates were harvested and a Western blot was performed to determine the

phosphorylation of S6RP (S240/244). -actin was used as a loading control. The bands

of the Western blot were quantified using NIH ImageJ. The phospho-bands were

normalized to the loading control and each condition is represented by average fold

change compared to the vehicle control. The images are from the same blot and

separated to present only the relevant treatment groups. The error bars represent the

SEM. The experiment was repeated a minimum of 3 times with similar results.

3

Fig. S3 The knockdown of raptor and rictor

ECs were transfected with either control, raptor or rictor siRNA. Seventy-two hours after

transfection the cell lysates were collected. A Western blot was performed to examine

the expression of raptor and rictor protein to determine the knockdown efficiency. Total

mTOR protein was used as a loading control. The bands were analyzed using NIH

ImageJ and normalized to mTOR. The percent expression was determined by

comparing the targets to the control siRNA.

4

Fig. S4 The knockdown of raptor and rictor using a second set of siRNAs

(a) ECs were transfected with either control, raptor or rictor siRNA. Seventy-two hours

after transfection the cell lysates were collected. A Western blot was performed to

examine the expression of raptor and rictor protein to determine the knockdown

efficiency. Total mTOR protein was used as a loading control. The bands were analyzed

using NIH ImageJ and normalized to mTOR. The percent expression was determined by

comparing the targets to the control siRNA. (b) EC were transfected with control, raptor

or rictor siRNA (an independent set compared to Figure 2). Twenty-four hours later a

fibrin sprouting assay (as described above) was performed. The beads were fixed at day

7 and imaged. The number of sprouts per bead was quantified. The error bars represent

the SEM (**p < 0.01). All experiments were repeated a minimum of 3 times with similar

results.

5

Figure S5. The knockdown of mTORC2 inhibits angiogenesis in vitro. (A and B) EC

were transfected with control, Rheb or Sin1 siRNA. Seventy-two hours after transfection

the cell lysates were collected. A Western blot was performed to examine the expression

of Rheb and Sin1 protein to determine the knockdown efficiency. -actin was used as a

loading control and the bands were analyzed using ImageJ. The percent expression was

determined by comparing the targets to the control siRNA.

6

Fig. S6 Rapamycin dose response

A western blot was performed on EC treated with a range of doses of rapamycin (as

indicated). The membrane was probed for S6RP (S240/244) and Akt (S473) to assess

for inhibition of mTORC1 and mTORC2, respectively. -actin was used as a loading

control. The bands of the Western blot were quantified using ImageJ. The phospho-

bands were normalized to the loading control and each condition is represented by

average fold change compared to the vehicle control. The error bars represent the SEM

(*p<0.05). All experiments were repeated a minimum of 3 times with similar results.

7

Fig. S7 Depletion of mTORC2 signaling by increasing the dose of rapamycin inhibits

sprouting in HMVEC

(a) HMVECs were coated onto Cytodex beads and embedded in a fibrin gel. NHLFs

were seeded on top of the fibrin gel. Twenty-four hours after embedding the cells were

treated with rapamycin as indicated. DMSO was used as a vehicle control. The inhibitor

was replaced with each medium change. (b) The beads were fixed at day 7 and the

number of sprouts per bead was quantified. Scale bar: 150 m. The error bars

represent the SEM (n = 2 independent experiments; ***p < 0.001).

8

Fig. S8 Pharmacological inhibition of mTOR does not reduce cell viability

EC we seeded into a 96-well plate and treated with vehicle, rapamycin (100nM) or

PP242 (600nM). After 48 hrs an XTT assay was performed and cell viability was

determined by measuring the OD at 450 nm. The results are presented at the percent

viability relative to the vehicle control. The error bars represent the SEM (n=3).

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Fig. S9 CXCR4 is co-expressed with EC in vivo

The CT-26 control tumors described in Figure 5 were sectioned and stained with an anti-

CD31 antibody (left panel) and an anti-CXCR4 antibody (middle panel). A merged image

(right panel) of the staining was obtained along with a DAPI nuclear stain (blue) to show

the co-expression of CD31 and CXCR4 (yellow). The fluorescent images of the sections

were taken at 40X and are representative of the staining observed in the control tumors.

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Fig. S10 High dose rapamycin blocks mTORC2 signaling and vessel infiltration in vivo

(a) EC within the tumor sections were visualized by staining for CD31+ cells. The

sections were imaged from one side of the tumor to the other to obtain the vascular

density for each image of the tumor as shown. (b) Using ImageJ, the degree of

vascularization was calculated at a relative distance into the tumor of 20%, 40%, 60%,

80% and 100% (where 100% was considered the center of the tumor). Error bars are the

SEM. n = 5-6 mice per group; (*p < 0.05).

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Fig. S11 PFKFB3 expression in EC. EC were transfected with control or PFKFB3

siRNA. Seventy-two hours after transfection the cell lysates were collected. A Western

blot was performed to examine the expression of PFKFB3 protein to determine the

knockdown efficiency. -actin was used as the loading control. The bands were

analyzed using ImageJ and the percent expression was determined by comparing the

targets to the control siRNA.

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