DIONESUS algorithm reveals new drug targets from dynamic phosphoproteomic networks

The DIONESUS algorithm provides scalable and
accurate reconstruction of dynamic phosphoproteomic
networks to reveal new drug targets: Supplement
Mark F. Ciaccio, Vincent C. Chen, Richard B. Jones, and Neda Bagheri
May 5, 2015
Extended Notes
1 Supplementary Notes 2
1.1 Phosphorylation kinetics, together with cell viability, inform modeling of informa-
tion flow downstream of EGFR. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1.1.1 Autocrine signaling alters carcinogenic pathways in A431 cells. . . . . . . . . 3
1.1.2 Distinct antioxidants demonstrate positive and negative influences on A431
cell viability. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
1.1.3 Reactive oxygen species affects cell receptor cross-phosphorylation. . . . . . 4
List of Figures
1 Viability as a function of EGF concentration in A431 cells . . . . . . . . . . . . . . . . 6
2 Serial graphs of phosphorylation kinetics for Panel 1 of growth factors . . . . . . . . 7
3 Serial graphs of phosphorylation kinetics for Panel 2 of small molecules . . . . . . . 13
8 Clustergram of phosphorylation kinetics for Panel 1 of growth factors . . . . . . . . 43
9 Clustergram of phosphorylation kinetics for Panel 2 of small molecules . . . . . . . 44
14 Volcano plots for all inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
15 Comparison of simple linear regression models . . . . . . . . . . . . . . . . . . . . . 64
16 Receiver operating characteristic (ROC) curves and precision-recall curves for two
in silico networks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
17 Inference of three node in silico networks using DIONESUS . . . . . . . . . . . . . . 66
18 Comparison of DIONESUS algorithm to GENIE3, Inferelator, and TIGRESS . . . . . 67
19 Quantification of microwestern bands. . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
1
Electronic Supplementary Material (ESI) for Integrative Biology.
This journal is © The Royal Society of Chemistry 2015

1 Supplementary Notes
1.1 Phosphorylation kinetics, together with cell viability, inform modeling of
information flow downstream of EGFR.
The first set of perturbations consisted of a panel of growth factors alone and in combination:
EGF (200 ng/mL), EGF (200 ng/ml) + Insulin (200 ng/mL), HGF (200 ng/mL), HGF (200
ng/mL) + insulin (200 ng/mL), insulin (200 ng/mL), IGF (200 ng/mL), and media alone control.
Phosphorylation abundances were measured at -30 min prior to stimulation, at the time of
stimulation (0 min), and 5 and 15 min following stimulation. The fold changes were assessed
by dividing each time point by the abundance at -30 min. For a positive control to measure
the specificity of stimulation, we quantified the conjugate receptor for each perturbant (EGFR
for EGF, c-MET for HGF, Insulin receptor for insulin, and IGFR receptor for IGF). Despite a
higher level of EGFR expression relative to the other RTKs, each stimulation showed a strong and
unique phosphorylation signature (Supplementary Figs. 2 and 8a). EGF stimulation preferentially
phosphorylated SRC-family kinase (SFK) substrates, STAT proteins, and PLCγ/calcium associated
proteins, but had little change in the AKT/survival pathway. IGF, HGF, and insulin contributed
strongly to the activation of the survival pathway. IGF caused an increased activation of p-S6
Ribosomal Protein and p-PKCG(PAN), both known to be important mediators of translational
regulation and cell growth.[1] We displayed the data from this set as a clustergram of fold
changes in phosphorylation (minus the media-alone results) to show the common and specific
subgroups that are regulated by each perturbation. EGF stimulation caused a strong reduction in
cell viability(35.1±3.9% of media control), Insulin and IGF caused a minor decrease (70.2±1.8%
and 78.9±0.8%), and HGF showed little change (92.0±9.4%) (Supplementary Fig. 8b). Only the
combination of HGF and insulin caused an increase in cell viability (110.5±1.5%) relative to the
media control, suggesting additivity of the survival signals downstream of these receptors. Insulin
in combination with EGF did not rescue the decrease in cell viability from EGF-alone (33.0±34.6%),
suggesting the predominance of negative mediators of cell viability downstream of EGFR after
stimulation with this concentration of growth factor.
We chose the first set of small-molecule inhibitors to block major pathways downstream of
the EGF receptor. As shown in the first experiment, the PLCγ proteins, the PI3K pathways, and
the SRC-family kinase pathways showed significant phosphorylation after EGF stimulation. The
secondary signaling molecules are known to be major binders of the phosphorylated tyrosines
on RTKs though their SH2- domains.[2] For these reasons, we hypothesized that these nodes
controlled major downstream mediators of the EGF signaling and that their specific inhibition
would elucidate the downstream pathways that contribute positively and negatively to cell viability.
To test this, we stimulated cells with 200 ng/mL EGF in conjunction with 10 µM PP2 (SRC Family
Kinase Inhibitor), 10 µM U73122 (PLCγ1/2 inhibitor), and 10 µM Wortmannin (PI3K inhibitor),
along with EGF-only and media-only controls. We added inhibitors 30 minutes before the
application of EGF to allow significant inhibitory activity to occur before stimulation and to assess
the effect of the inhibitor alone on phosphorylation. The time of application of the inhibitors was
designated as -30 mins, while time 0 mins was designated as the time of EGF application. We
measured additional time points at 5 and 15 mins after stimulation and assayed for changes in
phosphorylation by the MWA (Supplementary Figs. 3 and 9a).
We found that PP2 completely attenuated phosphorylation of p-STAT1(Y701), p-PYK(Y402),
p-P38(T180/Y182), and p-ERK(T202/Y204). It also decreased survival-associated phosphoproteins
such as p-AKT(S473). Both wortmannin and U73122 caused a major decrease in AKT substrates
p-GAB2(S159) and p-RAF(S338), and cell-cycle protein p-CDK2(T160). Surprisingly, there was
2

an increase in the amplitude and duration of p-P38(T180/Y182) and p-JNK(T183/Y182). While
200 ng/mL EGF with wortmannin, and EGF with U73122, caused a decrease in cell viability
(28.1±1.3% and 15.1±0.4%, respectively) in comparison to EGF-only (32.9±8.2%), application of
PP2 caused a complete attenuation of EGF-stimulated cell death (97.3±10.5%) (Supplementary
Fig. 9b).
1.1.1 Autocrine signaling alters carcinogenic pathways in A431 cells.
Autocrine signaling has been reported to cause cross-phosphorylation of receptor tyrosine kinases
in A431 cells.[3] A panel of sheddase inhibitors (aprotinin, GM6001, and TAPI1) also reported
to be effective in surveying autocrine signaling in A431 cells[4] was used in conjunction with
EGF stimulation. We hypothesized that sheddases could mediate the EGFR-induced RTK cross-
phosphorylation. 1 ug/mL aprotinin (a plasmin and general serine protease inhibitor), 25 µM
GM6001 (a broad-range metalloprotease inhibitor), and 100 µM TAPI1 (a TACE inhibitor) were
applied before 200 ng/mL EGF stimulation and the phosphorylation kinetics were assayed by
MWA (Supplementary Figs. 4 and 10a). Aprotinin showed a major increase in phosphorylation
of p-INS/IGF1R(Y1134/1135), p-PKCG(PAN)(T514), p-P70S6 Ribosomal Kinase(T421/S422), and
p-S6 Ribosomal Protein(S240/244) at 0 min. These proteins match the profile of those stimulated
by IGF, suggesting that aprotinin inhibits the cleavage of an IGF binding protein. GM6001
and TAPI1 showed a significant delay in p-INS/IGF1R(Y1134/1135) phosphorylation as well as
the reduction in phosphorylation of many known downstream mediators in the IGF-pathway
including p-AKT(S473), p-GAB2(S159), and p-RAF(S259). Aprotinin treatment did not cause a
change in cell viability (30.24±9.88%) in comparison to 200 ng/mL EGF alone (32.88±8.18%), while
GM6001 and TAPI1 showed almost complete loss of cell viability (12.25±8.25% and 2.48±2.48%)
(Supplementary Fig. 10b). Together, these results suggest that autocrine signaling is a key
mediator of cell viability in these cancer cell lines and that phosphorylation profiles of RTK and
downstream targets are modified by inhibition of these pathways. Autocrine signaling may be
one means by which a cancer cell overexpressing EGFR can activate multiple pathways leading to
carcinogenic behavior.
1.1.2 Distinct antioxidants demonstrate positive and negative influences on A431 cell viabil-
ity.
Reactive Oxygen Species (ROS) and its inhibition of phosphatase activity has been shown to be
important in EGF-induced RTK cross-phosphorylation in A431 cells.[5] To survey the contribution
of ROS, A431 cells were treated with the antioxidant panel, 100 µM Epigallactocatechin Gallate
(EGCG),10 µM Caffeic Acid Phenylethyl Ester (CAPE), and 30 mM N-acetylcysteine (N-ac)
30 minutes prior to EGF stimulation (Supplementary Figs. 5 and 11a). All antioxidants that
caused a common down-regulation of the PI3K/AKT pathway included p-INS/IGFR(Y1134/1135),
suggesting that much of the impact of ROS is mediated through this receptor. EGCG specifically
caused A431 cells to become insensitive to EGF stimulation. EGCG also caused significant and
specific increase in the phosphorylation of P38, JNK, and IGFR, with a total loss of cell viability
(Supplementary Fig. 11b). Since EGCG had zero viability and a phosphorylation profile divorced
from other perturbations, it was excluded from the final PLSR analysis. In addition to suppressing
the AKT-pathway, CAPE caused a specific decrease in the phosphorylation of p-CDK2(T160) with
a strong decrease in cell viability (3.60±3.20%). N-ac caused a decrease in the survival pathway but
an increase in the MEK/ERK pathway, resulting in a net increase in cell viability (58.21±15.15%) in
comparison to EGF alone (37.52±20.72%). In summary, antioxidants decreased the AKT pathway,
but they had variable effects on other cell pathways and increased or decreased viability of the
3

cancer cells, suggesting that antioxidant effect alone is probably insufficient as a general treatment
in this cancer model.
1.1.3 Reactive oxygen species affects cell receptor cross-phosphorylation.
Stimulation of A431 cells was reported to cause inactivation of protein-tyrosine phosphatase
1B through the release of reactive oxygen species.[6] We therefore chose the fourth and fifth
panels of inhibitors to study how ROS release affects cross-phosphorylation (Supplementary Figs.
6,7,12,13). Studies have shown expression of NADPH Oxidase in A431 cells and its stimulation
by EGF to produce ROS[5]. Therefore, two concentrations (10 µM and 25 µM) of the NADPH
oxidase inhibitor, Diphenyleneiodonium chloride (DPI), were used, along with an inhibitor of the
regulatory subunit of the NADPH oxidase complex, inhibitor of RAC1 (2 ng/mL Clostridium
Toxin B), and a PTP1B inhibitor, 25 µM EMBPTP1B1. As SHP2 is an SH2-domain containing
phosphatase relating to the highest fold change after EGF stimulation [7], the SHP2 inhibitor, at
20 µM PHPS1, was used after EGF stimulation to observe its contribution to phosphokinetics and
cell viability. Both concentrations of DPI showed no change in cell viability (10 µM = 31.69±9.74%;
25 µM = 26.35±3.85%) from EGF alone (29.36±3.77%). However, Clostridium Toxin demonstrated
a decrease in viability (15.29±2.98%). The PTP1B inhibitor resulted in a slight decrease in cell
viability (30.12±3.16%), whereas the SHP2 inhibitor showed a major decrease in cell viability
(4.50±4.17%) with a very large increase in the fold change of STAT1 phosphorylation, suggesting
that this phosphatase is a major mediator of the pro-apoptotic STAT1 phosphorylation and a
possible target in a subset of cancers. Finally, the cells were stimulated with the SRC-inhibitor, PP2,
at a lower concentration (200 nM) than in Inhibitor Set 1 to show greater specificity to the SRC
Family Kinases. Again, we confirmed that this condition increased cell viability (44.29±3.60%),
over EGF-stimulation alone, demonstrating that a subset of phosphosites downstream of Src-family
kinases negatively contribute to cell viability in this cancer model.
References
[1] Dufner, A. & Thomas, G. Ribosomal s6 kinase signaling and the control of transla-
tion. Experimental Cell Research 253, 100–109 (1999). URL http://www.sciencedirect.com/
science/article/pii/S0014482799946839.
[2] Jones, R. B., Gordus, A., Krall, J. A. & MacBeath, G. A quantitative protein interaction
network for the ErbB receptors using protein microarrays. Nature 439, 168ï£¡174 (2006). URL
http://www.ncbi.nlm.nih.gov/pubmed/16273093. PMID: 16273093.
[3] Shvartsman, S. Y. et al. Autocrine loops with positive feedback enable context-dependent cell
signaling. American Journal of Physiology - Cell Physiology 282, C545 –C559 (2002). URL
http://ajpcell.physiology.org/content/282/3/C545.abstract.
[4] Roztocil, E., Nicholl, S. M. & Davies, M. G. Insulin-induced epidermal growth factor activation
in vascular smooth muscle cells is ADAM-dependent. Surgery 144, 245–251 (2008). URL
http://www.sciencedirect.com/science/article/pii/S0039606008001864.
[5] Lou, Y. et al. Redox regulation of the protein tyrosine phosphatase PTP1B in cancer
cells. The FEBS Journal 275, 69â ˘A¸S88 (2008). URL http://www.ncbi.nlm.nih.gov/pubmed/
18067579. PMID: 18067579.
4

[6] Lee, S., Kwon, K., Kim, S. & Rhee, S. G. Reversible inactivation of Protein-Tyrosine phosphatase
1B in a431 cells stimulated with epidermal growth factor. Journal of Biological Chemistry 273,
15366–15372 (1998). URL http://www.jbc.org/content/273/25/15366.
[7] Ciaccio, M. F., Wagner, J. P., Chuu, C., Lauffenburger, D. A. & Jones, R. B. Systems analysis
of EGF receptor signaling dynamics with microwestern arrays. Nature Methods 7, 148–155
(2010). URL http://www.ncbi.nlm.nih.gov/pubmed/20101245. PMID: 20101245.
5

0
0.2
0.4
0.6
0.8
1
1.2
1.4Relativecellviability(Mediaalone=1.0) Cell viability as a function of EGF concentrationM
ediaw
ith
8%
Serum
M
ediaalone0.2
ng/m
LEGF
2
ng/m
LEGF50
ng/m
LEGF100
ng/m
LEGF200
ng/m
LEGF
Figure 1: Viability as a function of EGF concentration in A431 cells. Low concentrations increase cell viability in
A431 cells after 24 hrs relative to media-alone stimulation while high concentrations of EGF induce a loss of
cell viability.
.
6

Stimulation of A431 Cells with a Panel of Growth Factors:
Media Change at −30 min, Growth Factor w/ Media added at 0 min
−4 −2 0 2 4 6 8 10 12 14 16
0
1
2
3
4
5
6
p−ERK(T202/Y204)
Time after Growth Factor Stimulation (min)
FoldChangeinPhosphorylationfrom−30min
Serum−free Media(DMEM)
EGF(100 ng/mL)
EGF(100 ng/mL) + Insulin(200 ng/mL)
HGF(200 ng/mL)
HGF(200 ng/mL) + Insulin(200 ng/mL)
Insulin(200 ng/mL)
IGF(200 ng/mL)
Figure 2: Serial graphs of phosphorylation kinetics for Panel 1 of growth factors. Subsequent serial graphs are shown
on the next 5 pages, where the x-axis reflects time (in minutes) and the y-axis reflects fold change relative to
-30 minutes (A fold change of 1 reflects no change from -30 minutes)
.
7

−5 0 5 10 15
0
1
2
3
p−4EBP1(S65)
−5 0 5 10 15
0
0.5
1
1.5
p−4EBP1(T37/46)
−5 0 5 10 15
0
0.5
1
1.5
p−ABL(C−)(Y245)
−5 0 5 10 15
0
2
4
6
8
p−AKT(S473)
−5 0 5 10 15
0
1
2
p−CDC2(Y15)
−5 0 5 10 15
0
2
4
6
p−CDK2(T160)
−5 0 5 10 15
0
1
2
3
p−CREB(S133)
−5 0 5 10 15
0
2
4
6
p−CRKII(Y221)
−5 0 5 10 15
0
1
2
p−CRKL(Y207)
−5 0 5 10 15
0
2
4
6
8
p−EGFR(Y1068)
−5 0 5 10 15
0
10
20
p−EGFR(Y1086)
−5 0 5 10 15
0
5
10
p−EGFR(Y1173)
−5 0 5 10 15
0
5
10
p−EGFR(Y845)
−5 0 5 10 15
0
1
2
3
p−ERBB2(Y1221/1222)
−5 0 5 10 15
0
1
2
p−ERBB4(Y1284)

−5 0 5 10 15
0
2
4
p−ERK(T202/Y204)
−5 0 5 10 15
0
1
2
p−FAK(Y576/577)
−5 0 5 10 15
0
2
4
p−FGFR1(Y653/654)
−5 0 5 10 15
0
0.5
1
1.5
p−FOXO1(S319)/FOXO4(S262)
−5 0 5 10 15
0
1
2
3
p−FOXO3A(S253)
−5 0 5 10 15
0
2
4
p−GAB1(Y627)
−5 0 5 10 15
0
2
4
6
p−GAB2(S159)
−5 0 5 10 15
0
2
4
6
p−GSK3A(S21)
−5 0 5 10 15
0
10
20
p−IGF1RB (Y1135/1136) /p−InsR…
−5 0 5 10 15
0
2
4
p−IRS1(S1101)
−5 0 5 10 15
0
2
4
p−JAK1(Y1022/1023)
−5 0 5 10 15
0
1
2
p−KIT(C−)(Y719)
−5 0 5 10 15
0
1
2
3
p−MEK(1/2)(S217/221)
−5 0 5 10 15
0
5
10
p−MET(Y1234/1235)
−5 0 5 10 15
0
2
4
p−MET(Y1349)

−5 0 5 10 15
0
1
2
p−MTOR(S2448)
−5 0 5 10 15
0
1
2
p−P38MAPK(T180/Y182)
−5 0 5 10 15
0
1
2
p−P70S6K(T421/S424)
−5 0 5 10 15
0
2
4
p−P90RSK(S380)
−5 0 5 10 15
0
2
4
6
p−PDFGRA(Y849)/PDGFRB(Y857)
−5 0 5 10 15
0
2
4
p−PDGFRA(Y754)
−5 0 5 10 15
0
2
4
p−PDGFRB(Y1009)
−5 0 5 10 15
0
2
4
p−PDK1(S241)
−5 0 5 10 15
0
1
2
p−PI3KP85(Y458)P55(Y199)
−5 0 5 10 15
0
2
4
p−PKCD(T505)
−5 0 5 10 15
0
2
4
6
p−PKCPAN/G(T514)
−5 0 5 10 15
0
2
4
6
8
p−PLCG1(Y783)
−5 0 5 10 15
0
2
4
6
8
p−PLCG2(Y759)
−5 0 5 10 15
0
1
2
p−PTEN(S380)
−5 0 5 10 15
0
1
2
3
p−PYK2(Y402)

−5 0 5 10 15
0
1
2
3
p−RAF(C−)(S259)
−5 0 5 10 15
0
2
4
6
p−RAF(C−)(S338)
−5 0 5 10 15
0
2
4
6
p−S6RIBPROT(S240/244)
−5 0 5 10 15
0
1
2
p−SAPK/JNK(T183/Y185)
−5 0 5 10 15
0
1
2
p−SHC(Y239/240)
−5 0 5 10 15
0
1
2
3
p−SHP2(Y542)
−5 0 5 10 15
0
0.5
1
1.5
p−SRC(Y416)
−5 0 5 10 15
0
0.5
1
1.5
p−SRC(Y527)
−5 0 5 10 15
0
1
2
p−STAT1(Y701)
−5 0 5 10 15
0
1
2
p−STAT3(S727)
−5 0 5 10 15
0
1
2
3
p−STAT3(Y705)
−5 0 5 10 15
0
1
2
3
p−STAT5(Y694)
−5 0 5 10 15
0
2
4
p−STAT6(Y641)
−5 0 5 10 15
0
2
4
p−SYK(Y525/526)
−5 0 5 10 15
0
5
10
p−ZAP70(Y319)/SYK(Y352)

−5 0 5 10 15
0
0.5
1
1.5
ACTIN(SET1)
−5 0 5 10 15
0
0.5
1
1.5
ACTIN(SET2)
−5 0 5 10 15
0
1
2
GAPDH(SET1)
−5 0 5 10 15
0
1
2
GAPDH(SET2)
−5 0 5 10 15
0
0.5
1
1.5
TUBULIN(SET1)
−5 0 5 10 15
0
0.5
1
1.5
TUBULIN(SET2)

Stimulation of A431 Cells with a Panel of Small Molecules (X2):
−4 −2 0 2 4 6 8 10 12 14 16
0
0.5
1
1.5
2
2.5
3
3.5
EGF(200 ng/mL)
EGF(200 ng/mL)+PP2 (10 uM)
EGF(200 ng/mL)+U73122 (10 uM)
EGF(200 ng/mL)+Wortmannin (10 uM)
Figure 3: Serial graphs of phosphorylation kinetics for Panel 2 of small molecules. Subsequent serial graphs are shown
-30 minutes (A fold change of 1 reﬂects no change from -30 minutes).
13

−5 0 5 10 15
0
1
2
p−4EBP1(S65)
−5 0 5 10 15
0
1
2
3
p−4EBP1(T37/46)
−5 0 5 10 15
0
0.5
1
1.5
p−ABL(C−)(Y245)
−5 0 5 10 15
0
2
4
p−AKT(S473)
−5 0 5 10 15
0
2
4
p−CDC2(Y15)
−5 0 5 10 15
0
1
2
p−CDK2(T160)
−5 0 5 10 15
0
1
2
p−CREB(S133)
−5 0 5 10 15
0
0.5
1
1.5
p−CRKII(Y221)
−5 0 5 10 15
0
1
2
p−CRKL(Y207)
−5 0 5 10 15
0
20
40
p−EGFR(Y1068)
−5 0 5 10 15
0
20
40
60
80
p−EGFR(Y1086)
−5 0 5 10 15
0
5
10
p−EGFR(Y1173)
−5 0 5 10 15
0
2
4
6
8
p−EGFR(Y845)
−5 0 5 10 15
0
1
2
p−ERBB2(Y1221/1222)
−5 0 5 10 15
0
1
2
p−ERBB4(Y1284)

−5 0 5 10 15
0
5
10
p−ERK(T202/Y204)
−5 0 5 10 15
0
1
2
p−FAK(Y576/577)
−5 0 5 10 15
0
2
4
6
8
p−FGFR1(Y653/654)
−5 0 5 10 15
0
0.5
1
1.5
−5 0 5 10 15
0
2
4
p−FOXO3A(S253)
−5 0 5 10 15
0
2
4
p−GAB1(Y627)
−5 0 5 10 15
0
1
2
3
p−GAB2(S159)
−5 0 5 10 15
0
1
2
3
p−GSK3A(S21)
−5 0 5 10 15
0
1
2
3
−5 0 5 10 15
0
1
2
p−IRS1(S1101)
−5 0 5 10 15
0
2
4
p−JAK1(Y1022/1023)
−5 0 5 10 15
0
2
4
p−KIT(C−)(Y719)
−5 0 5 10 15
0
2
4
p−MEK(1/2)(S217/221)
−5 0 5 10 15
0
5
10
p−MET(Y1234/1235)
−5 0 5 10 15
0
10
20
30
p−MET(Y1349)

−5 0 5 10 15
0
1
2
3
p−MTOR(S2448)
−5 0 5 10 15
0
2
4
6
−5 0 5 10 15
0
1
2
p−P70S6K(T421/S424)
−5 0 5 10 15
0
1
2
p−P90RSK(S380)
−5 0 5 10 15
0
0.5
1
1.5
−5 0 5 10 15
0
2
4
6
p−PDGFRA(Y754)
−5 0 5 10 15
0
2
4
p−PDGFRB(Y1009)
−5 0 5 10 15
0
1
2
p−PDK1(S241)
−5 0 5 10 15
0
1
2
3
p−PI3KP85(Y458)P55(Y199)
−5 0 5 10 15
0
0.5
1
1.5
p−PKCD(T505)
−5 0 5 10 15
0
1
2
p−PKCPAN/G(T514)
−5 0 5 10 15
0
2
4
6
8
p−PLCG1(Y783)
−5 0 5 10 15
0
2
4
6
8
p−PLCG2(Y759)
−5 0 5 10 15
0
1
2
p−PTEN(S380)
−5 0 5 10 15
0
2
4
6
p−PYK2(Y402)

−5 0 5 10 15
0
1
2
3
p−RAF(C−)(S259)
−5 0 5 10 15
0
0.5
1
1.5
p−RAF(C−)(S338)
−5 0 5 10 15
0
1
2
3
−5 0 5 10 15
0
1
2
−5 0 5 10 15
0
2
4
p−SHC(Y239/240)
−5 0 5 10 15
0
2
4
6
8
p−SHP2(Y542)
−5 0 5 10 15
0
5
10
15
p−SRC(Y416)
−5 0 5 10 15
0
1
2
p−SRC(Y527)
−5 0 5 10 15
0
5
10
p−STAT1(Y701)
−5 0 5 10 15
0
1
2
p−STAT3(S727)
−5 0 5 10 15
0
2
4
p−STAT3(Y705)
−5 0 5 10 15
0
1
2
p−STAT5(Y694)
−5 0 5 10 15
0
2
4
6
8
p−STAT6(Y641)
−5 0 5 10 15
0
5
10
p−SYK(Y525/526)
−5 0 5 10 15
0
2
4
p−ZAP70(Y319)/SYK(Y352)

−5 0 5 10 15
0
0.5
1
1.5
ACTIN(SET1)
−5 0 5 10 15
0
0.5
1
1.5
ACTIN(SET2)
−5 0 5 10 15
0
0.5
1
1.5
TUBULIN(SET1)
−5 0 5 10 15
0
0.5
1
1.5
TUBULIN(SET2)
−5 0 5 10 15
0
0.5
1
1.5
GAPDH(SET1)
−5 0 5 10 15
0
0.5
1
1.5
GAPDH(SET2)

Stimulation of A431 Cells with a Panel of Protease Inhibitors (X3):
−4 −2 0 2 4 6 8 10 12 14 16
5
10
15
20
25
EGF(200 ng/mL)
EGF(200 ng/mL)+Aprotinin(1 ug/mL)
EGF(200 ng/mL)+GM6001(25 uM)
EGF(200 ng/mL)+TAPI1(100 uM)
19

−5 0 5 10 15
0
1
2
3
p−4EBP1(S65)
−5 0 5 10 15
0
1
2
p−4EBP1(T37/46)
−5 0 5 10 15
0
1
2
3
p−ABL(C−)(Y245)
−5 0 5 10 15
0
5
10
15
p−AKT(S473)
−5 0 5 10 15
0
2
4
p−CDC2(Y15)
−5 0 5 10 15
0
2
4
6
8
p−CDK2(T160)
−5 0 5 10 15
0
2
4
p−CREB(S133)
−5 0 5 10 15
0
5
10
15
p−CRKII(Y221)
−5 0 5 10 15
0
1
2
3
p−CRKL(Y207)
−5 0 5 10 15
0
5
10
p−EGFR(Y1068)
−5 0 5 10 15
0
20
40
60
p−EGFR(Y1086)
−5 0 5 10 15
0
5
10
15
p−EGFR(Y1173)
−5 0 5 10 15
0
5
10
p−EGFR(Y845)
−5 0 5 10 15
0
10
20
p−ERBB2(Y1221/1222)
−5 0 5 10 15
0
2
4
6
p−ERBB4(Y1284)

−5 0 5 10 15
0
2
4
p−ERK(T202/Y204)
−5 0 5 10 15
0
5
10
15
p−FAK(Y576/577)
−5 0 5 10 15
0
5
10
p−FGFR1(Y653/654)
−5 0 5 10 15
0
1
2
3
−5 0 5 10 15
0
2
4
p−FOXO3A(S253)
−5 0 5 10 15
0
2
4
6
p−GAB1(Y627)
−5 0 5 10 15
0
1
2
p−GAB2(S159)
−5 0 5 10 15
0
2
4
p−GSK3A(S21)
−5 0 5 10 15
0
2
4
6
8
−5 0 5 10 15
0
1
2
p−IRS1(S1101)
−5 0 5 10 15
0
5
10
p−JAK1(Y1022/1023)
−5 0 5 10 15
0
0.5
1
1.5
p−KIT(C−)(Y719)
−5 0 5 10 15
0
2
4
p−MEK(1/2)(S217/221)
−5 0 5 10 15
0
10
20
p−MET(Y1234/1235)
−5 0 5 10 15
0
2
4
p−MET(Y1349)

−5 0 5 10 15
0
1
2
3
p−MTOR(S2448)
−5 0 5 10 15
0
2
4
6
−5 0 5 10 15
0
2
4
p−P70S6K(T421/S424)
−5 0 5 10 15
0
2
4
p−P90RSK(S380)
−5 0 5 10 15
0
10
20
30
−5 0 5 10 15
0
2
4
p−PDGFRA(Y754)
−5 0 5 10 15
0
5
10
p−PDGFRB(Y1009)
−5 0 5 10 15
0
2
4
p−PDK1(S241)
−5 0 5 10 15
0
2
4
6
p−PI3KP85(Y458)P55(Y199)
−5 0 5 10 15
0
5
10
p−PKCD(T505)
−5 0 5 10 15
0
10
20
p−PKCPAN/G(T514)
−5 0 5 10 15
0
5
10
15
p−PLCG1(Y783)
−5 0 5 10 15
0
5
10
15
p−PLCG2(Y759)
−5 0 5 10 15
0
1
2
p−PTEN(S380)
−5 0 5 10 15
0
5
10
p−PYK2(Y402)

−5 0 5 10 15
0
2
4
p−RAF(C−)(S259)
−5 0 5 10 15
0
5
10
p−RAF(C−)(S338)
−5 0 5 10 15
0
10
20
−5 0 5 10 15
0
1
2
−5 0 5 10 15
0
1
2
3
p−SHC(Y239/240)
−5 0 5 10 15
0
5
10
15
p−SHP2(Y542)
−5 0 5 10 15
0
5
10
15
p−SRC(Y416)
−5 0 5 10 15
0
1
2
p−SRC(Y527)
−5 0 5 10 15
0
10
20
p−STAT1(Y701)
−5 0 5 10 15
0
1
2
3
p−STAT3(S727)
−5 0 5 10 15
0
2
4
6
8
p−STAT3(Y705)
−5 0 5 10 15
0
0.5
1
1.5
p−STAT5(Y694)
−5 0 5 10 15
0
20
40
p−STAT6(Y641)
−5 0 5 10 15
0
10
20
30
p−SYK(Y525/526)
−5 0 5 10 15
0
5
10
15
p−ZAP70(Y319)/SYK(Y352)

−5 0 5 10 15
0
0.5
1
1.5
ACTIN(SET1)
−5 0 5 10 15
0
1
2
ACTIN(SET2)
−5 0 5 10 15
0
0.5
1
1.5
GAPDH(SET1)
−5 0 5 10 15
0
0.5
1
1.5
GAPDH(SET2)
−5 0 5 10 15
0
0.5
1
1.5
TUBULIN(SET1)
−5 0 5 10 15
0
1
2
TUBULIN(SET2)

−4 −2 0 2 4 6 8 10 12 14 16
2
4
6
8
10
12
14
16
18
p−AKT(S473)
EGF(200 ng/mL)
EGF(200 ng/mL)+CAPE(10 uM)
EGF(200 ng/mL)+EGCG(100 uM)
EGF(200 ng/mL)+N−Acetylcysteine(30 mM)
25

−5 0 5 10 15
0
0.5
1
1.5
p−4EBP1(S65)
−5 0 5 10 15
0
1
2
3
p−4EBP1(T37/46)
−5 0 5 10 15
0
0.5
1
1.5
p−ABL(C−)(Y245)
−5 0 5 10 15
0
5
10
15
p−AKT(S473)
−5 0 5 10 15
0
2
4
6
p−CDC2(Y15)
−5 0 5 10 15
0
2
4
6
8
p−CDK2(T160)
−5 0 5 10 15
0
2
4
p−CREB(S133)
−5 0 5 10 15
0
2
4
p−CRKII(Y221)
−5 0 5 10 15
0
1
2
3
p−CRKL(Y207)
−5 0 5 10 15
0
2
4
6
p−EGFR(Y1068)
−5 0 5 10 15
0
10
20
30
p−EGFR(Y1086)
−5 0 5 10 15
0
2
4
6
p−EGFR(Y1173)
−5 0 5 10 15
0
2
4
p−EGFR(Y845)
−5 0 5 10 15
0
2
4
p−ERBB2(Y1221/1222)
−5 0 5 10 15
0
1
2
3
p−ERBB4(Y1284)

−5 0 5 10 15
0
5
10
p−ERK(T202/Y204)
−5 0 5 10 15
0
2
4
6
p−FAK(Y576/577)
−5 0 5 10 15
0
2
4
p−FGFR1(Y653/654)
−5 0 5 10 15
0
1
2
3
−5 0 5 10 15
0
2
4
p−FOXO3A(S253)
−5 0 5 10 15
0
2
4
6
p−GAB1(Y627)
−5 0 5 10 15
0
1
2
p−GAB2(S159)
−5 0 5 10 15
0
1
2
p−GSK3A(S21)
−5 0 5 10 15
0
5
10
−5 0 5 10 15
0
2
4
p−IRS1(S1101)
−5 0 5 10 15
0
2
4
p−JAK1(Y1022/1023)
−5 0 5 10 15
0
2
4
p−KIT(C−)(Y719)
−5 0 5 10 15
0
5
10
p−MEK(1/2)(S217/221)
−5 0 5 10 15
0
2
4
6
p−MET(Y1234/1235)
−5 0 5 10 15
0
1
2
3
p−MET(Y1349)

−5 0 5 10 15
0
2
4
p−MTOR(S2448)
−5 0 5 10 15
0
20
40
−5 0 5 10 15
0
1
2
p−P70S6K(T421/S424)
−5 0 5 10 15
0
2
4
6
p−P90RSK(S380)
−5 0 5 10 15
0
10
20
−5 0 5 10 15
0
2
4
6
p−PDGFRA(Y754)
−5 0 5 10 15
0
5
10
15
p−PDGFRB(Y1009)
−5 0 5 10 15
0
2
4
p−PDK1(S241)
−5 0 5 10 15
0
2
4
6
8
p−PI3KP85(Y458)P55(Y199)
−5 0 5 10 15
0
2
4
6
p−PKCD(T505)
−5 0 5 10 15
0
5
10
15
p−PKCPAN/G(T514)
−5 0 5 10 15
0
2
4
6
p−PLCG1(Y783)
−5 0 5 10 15
0
5
10
p−PLCG2(Y759)
−5 0 5 10 15
0
2
4
6
p−PTEN(S380)
−5 0 5 10 15
0
2
4
6
8
p−PYK2(Y402)

−5 0 5 10 15
0
1
2
p−RAF(C−)(S259)
−5 0 5 10 15
0
2
4
6
p−RAF(C−)(S338)
−5 0 5 10 15
0
5
10
15
−5 0 5 10 15
0
5
10
15
−5 0 5 10 15
0
5
10
p−SHC(Y239/240)
−5 0 5 10 15
0
10
20
30
p−SHP2(Y542)
−5 0 5 10 15
0
2
4
p−SRC(Y416)
−5 0 5 10 15
0
2
4
p−SRC(Y527)
−5 0 5 10 15
0
5
10
p−STAT1(Y701)
−5 0 5 10 15
0
2
4
6
p−STAT3(S727)
−5 0 5 10 15
0
0.5
1
1.5
p−STAT3(Y705)
−5 0 5 10 15
0
1
2
3
p−STAT5(Y694)
−5 0 5 10 15
0
10
20
p−STAT6(Y641)
−5 0 5 10 15
0
2
4
6
p−SYK(Y525/526)
−5 0 5 10 15
0
2
4
6
p−ZAP70(Y319)/SYK(Y352)

−5 0 5 10 15
0
0.5
1
1.5
ACTIN(SET1)
−5 0 5 10 15
0
0.5
1
1.5
ACTIN(SET2)
−5 0 5 10 15
0
1
2
GAPDH(SET1)
−5 0 5 10 15
0
1
2
GAPDH(SET2)
−5 0 5 10 15
0
1
2
3
TUBULIN(SET1)
−5 0 5 10 15
0
0.5
1
1.5
TUBULIN(SET2)

−4 −2 0 2 4 6 8 10 12 14 16
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
EGF(200 ng/mL)
EGF(200 ng/mL)+DPI(25 uM)
EGF(200 ng/mL)+Clostridium Toxin B(2 ng/mL)
EGF(200 ng/mL)+PHPS2(20 uM)
31

−5 0 5 10 15
0
2
4
6
8
p−4EBP1(S65)
−5 0 5 10 15
0
2
4
p−4EBP1(T37/46)
−5 0 5 10 15
0
2
4
p−ABL(C−)(Y245)
−5 0 5 10 15
0
1
2
3
p−AKT(S473)
−5 0 5 10 15
0
2
4
6
p−CDC2(Y15)
−5 0 5 10 15
0
0.5
1
1.5
p−CDK2(T160)
−5 0 5 10 15
0
1
2
p−CREB(S133)
−5 0 5 10 15
0
2
4
6
8
p−CRKII(Y221)
−5 0 5 10 15
0
1
2
p−CRKL(Y207)
−5 0 5 10 15
0
10
20
p−EGFR(Y1068)
−5 0 5 10 15
0
50
100
p−EGFR(Y1086)
−5 0 5 10 15
0
5
10
p−EGFR(Y1173)
−5 0 5 10 15
0
2
4
6
p−EGFR(Y845)
−5 0 5 10 15
0
5
10
15
p−ERBB2(Y1221/1222)
−5 0 5 10 15
0
2
4
6
p−ERBB4(Y1284)

−5 0 5 10 15
0
5
10
p−ERK(T202/Y204)
−5 0 5 10 15
0
10
20
p−FAK(Y576/577)
−5 0 5 10 15
0
10
20
p−FGFR1(Y653/654)
−5 0 5 10 15
0
0.5
1
1.5
−5 0 5 10 15
0
1
2
3
p−FOXO3A(S253)
−5 0 5 10 15
0
2
4
6
p−GAB1(Y627)
−5 0 5 10 15
0
1
2
p−GAB2(S159)
−5 0 5 10 15
0
2
4
p−GSK3A(S21)
−5 0 5 10 15
0
2
4
6
8
−5 0 5 10 15
0
1
2
3
p−IRS1(S1101)
−5 0 5 10 15
0
5
10
15
p−JAK1(Y1022/1023)
−5 0 5 10 15
0
5
10
p−KIT(C−)(Y719)
−5 0 5 10 15
0
2
4
6
p−MEK(1/2)(S217/221)
−5 0 5 10 15
0
5
10
15
p−MET(Y1234/1235)
−5 0 5 10 15
0
2
4
6
p−MET(Y1349)

−5 0 5 10 15
0
1
2
3
p−MTOR(S2448)
−5 0 5 10 15
0
2
4
−5 0 5 10 15
0
1
2
3
p−P70S6K(T421/S424)
−5 0 5 10 15
0
2
4
p−P90RSK(S380)
−5 0 5 10 15
0
20
40
−5 0 5 10 15
0
5
10
p−PDGFRA(Y754)
−5 0 5 10 15
0
2
4
p−PDGFRB(Y1009)
−5 0 5 10 15
0
2
4
p−PDK1(S241)
−5 0 5 10 15
0
1
2
p−PI3KP85(Y458)P55(Y199)
−5 0 5 10 15
0
2
4
6
p−PKCD(T505)
−5 0 5 10 15
0
0.5
1
1.5
p−PKCPAN/G(T514)
−5 0 5 10 15
0
2
4
6
p−PLCG1(Y783)
−5 0 5 10 15
0
10
20
30
p−PLCG2(Y759)
−5 0 5 10 15
0
1
2
p−PTEN(S380)
−5 0 5 10 15
0
5
10
p−PYK2(Y402)

−5 0 5 10 15
0
2
4
p−RAF(C−)(S259)
−5 0 5 10 15
0
5
10
p−RAF(C−)(S338)
−5 0 5 10 15
0
1
2
3
−5 0 5 10 15
0
1
2
3
−5 0 5 10 15
0
1
2
3
p−SHC(Y239/240)
−5 0 5 10 15
0
10
20
p−SHP2(Y542)
−5 0 5 10 15
0
5
10
p−SRC(Y416)
−5 0 5 10 15
0
1
2
p−SRC(Y527)
−5 0 5 10 15
0
20
40
p−STAT1(Y701)
−5 0 5 10 15
0
1
2
p−STAT3(S727)
−5 0 5 10 15
0
2
4
6
8
p−STAT3(Y705)
−5 0 5 10 15
0
5
10
p−STAT5(Y694)
−5 0 5 10 15
0
20
40
p−STAT6(Y641)
−5 0 5 10 15
0
5
10
p−SYK(Y525/526)
−5 0 5 10 15
0
10
20
p−ZAP70(Y319)/SYK(Y352)

−5 0 5 10 15
0
2
4
ACTIN(SET1)
−5 0 5 10 15
0
1
2
ACTIN(SET2)
−5 0 5 10 15
0
1
2
3
GAPDH(SET1)
−5 0 5 10 15
0
0.5
1
1.5
GAPDH(SET2)
−5 0 5 10 15
0
0.5
1
1.5
TUBULIN(SET1)
−5 0 5 10 15
0
1
2
TUBULIN(SET2)

−4 −2 0 2 4 6 8 10 12 14 16
2
4
6
8
10
12
p−STAT1(Y701)
EGF(200 ng/mL)
EGF(200 ng/mL)+DPI(10 uM)
EGF(200 ng/mL)+PP2(200 nM)
EGF(200 ng/mL)+EMBPTP1BI(25 uM)
37

EGF200(0m)X1
EGF200(5m)X1
EGF200(15m)X1
EGF200+Insulin200(0m)X1
HGF200(0m)X1
HGF200(5m)X1
HGF200(15m)X1
HGF200+Insulin200(0m)X1
Insulin200(0m)X1
Insulin200(5m)X1
Insulin200(15m)X1
IGF200(0m)X1
IGF200(5m)X1
IGF200(15m)X1
p−MEK(1/2)(S217/221)
p−ERK(T202/Y204)
p−AKT(S473)
p−RAF(C−)(S338)
p−GSK3A(S21)
p−FOXO3A(S253)
p−PDK1(S241)
p−RAF(C−)(S259)
p−GAB2(S159)
p−IGF1RB (Y1135/1136) /…
p−ZAP70(Y319)/SYK(Y352)
p−CRKII(Y221)
p−STAT6(Y641)
p−MET(Y1349)
p−JAK1(Y1022/1023)
p−PDGFRA(Y754)
p−PKCD(T505)
p−STAT3(S727)
p−IRS1(S1101)
p−SHC(Y239/240)
p−ERBB2(Y1221/1222)
p−MTOR(S2448)
p−CDC2(Y15)
p−FOXO1(S319)/…
p−SYK(Y525/526)
p−FAK(Y576/577)
p−STAT3(Y705)
p−STAT1(Y701)
p−ERBB4(Y1284)
p−SRC(Y416)
p−ABL(C−)(Y245)
p−4EBP1(S65)
p−4EBP1(T37/46)
p−PTEN(S380)
p−SRC(Y527)
p−CRKL(Y207)
p−CREB(S133)
p−P70S6K(T421/S424)
p−PI3KP85(Y458)P55(Y199)
p−CDK2(T160)
p−P90RSK(S380)
p−PKCPAN/G(T514)
p−GAB1(Y627)
p−SHP2(Y542)
p−STAT5(Y694)
p−FGFR1(Y653/654)
p−PDGFRB(Y1009)
p−PYK2(Y402)
p−KIT(C−)(Y719)
p−PDFGRA(Y849)/…
p−EGFR(Y1068)
p−EGFR(Y1086)
p−PLCG2(Y759)
p−PLCG1(Y783)
p−MET(Y1234/1235)
p−EGFR(Y845)
p−EGFR(Y1173)
a)
b)
log(Fold Change from Time -30 min)
0 1 2-1-2
0 0.2 0.4 0.6 0.8 1 1.2 1.4
Media
EGF(200ng/mL)
EGF(200ng/mL)+Ins(200ng/mL)
HGF(200ng/mL)
HGF(200ng/mL)+Ins(200ng/mL)
Ins(200ng/mL)
IGF(200ng/mL)
Cell Viability as Fraction of Media alone
Figure 8: (a) The clustergram of phosphorylation kinetics is shown for Panel 1 of growth factors. (b) Relative cell
viability is shown after 24 hours perturbation, in comparison to media alone.
43

EGF200(0m)X2
EGF200(5m)X2
EGF200(15m)X2
EGF200PP2(0m)X2
EGF200PP2(5m)X2
EGF200PP2(15m)X2
EGF200U73122(0m)X2
EGF200U73122(5m)X2
EGF200U73122(15m)X2
EGF200Wortmannin(0m)X2
p−AKT(S473)
p−4EBP1(T37/46)
p−4EBP1(S65)
p−GSK3A(S21)
p−CREB(S133)
p−CRKL(Y207)
p−STAT3(S727)
p−FOXO1(S319)/…
p−PTEN(S380)
p−P70S6K(T421/S424)
p−P90RSK(S380)
p−SRC(Y527)
p−GAB2(S159)
p−PKCPAN/G(T514)
p−CDK2(T160)
p−PDK1(S241)
p−ABL(C−)(Y245)
p−PKCD(T505)
p−CRKII(Y221)
p−RAF(C−)(S338)
p−ERBB4(Y1284)
p−FOXO3A(S253)
p−FAK(Y576/577)
p−RAF(C−)(S259)
p−IRS1(S1101)
p−PI3KP85(Y458)P55(Y199)
p−JAK1(Y1022/1023)
p−STAT3(Y705)
p−MTOR(S2448)
p−STAT5(Y694)
p−ERBB2(Y1221/1222)
p−KIT(C−)(Y719)
p−IGF1RB (Y1135/1136) /…
p−CDC2(Y15)
p−SHC(Y239/240)
p−PDGFRB(Y1009)
p−PYK2(Y402)
p−GAB1(Y627)
p−ERK(T202/Y204)
p−MEK(1/2)(S217/221)
p−MET(Y1349)
p−ZAP70(Y319)/SYK(Y352)
p−PDGFRA(Y754)
p−SYK(Y525/526)
p−PLCG1(Y783)
p−SHP2(Y542)
p−FGFR1(Y653/654)
p−PLCG2(Y759)
p−STAT6(Y641)
p−STAT1(Y701)
p−EGFR(Y845)
p−SRC(Y416)
p−MET(Y1234/1235)
p−EGFR(Y1173)
p−EGFR(Y1068)
p−EGFR(Y1086)
0 1 2-1-2
0 0.5 1 1.5
Media
EGF(200ng/mL)
EGF(200ng/mL)+PP2(10uM)
EGF(200ng/mL)+U73122(10uM)
EGF(200ng/mL)+Wortmannin(10uM)
a)
b)
Figure 9: (a) The clustergram of phosphorylation kinetics is shown for Panel 2 of small molecules. (b) Relative cell
44

EGF200(0m)X3
EGF200(5m)X3
EGF200(15m)X3
EGF200Aprotinin(0m)X3
EGF200GM6001(0m)X3
EGF200GM6001(5m)X3
EGF200GM6001(15m)X3
EGF200TAPI−1(0m)X3
p−PDK1(S241)
p−PKCPAN/G(T514)
p−AKT(S473)
p−CDK2(T160)
p−P70S6K(T421/S424)
p−FOXO1(S319)/…
p−PDGFRA(Y754)
p−CRKII(Y221)
p−4EBP1(S65)
p−ERK(T202/Y204)
p−CRKL(Y207)
p−FOXO3A(S253)
p−MTOR(S2448)
p−GSK3A(S21)
p−CDC2(Y15)
p−SHC(Y239/240)
p−STAT3(S727)
p−IRS1(S1101)
p−ABL(C−)(Y245)
p−KIT(C−)(Y719)
p−STAT5(Y694)
p−SRC(Y527)
p−4EBP1(T37/46)
p−PTEN(S380)
p−GAB2(S159)
p−RAF(C−)(S259)
p−P90RSK(S380)
p−RAF(C−)(S338)
p−CREB(S133)
p−ERBB4(Y1284)
p−PKCD(T505)
p−PI3KP85(Y458)P55(Y199)
p−GAB1(Y627)
p−MEK(1/2)(S217/221)
p−MET(Y1349)
p−SHP2(Y542)
p−IGF1RB (Y1135/1136) /…
p−ZAP70(Y319)/SYK(Y352)
p−STAT3(Y705)
p−JAK1(Y1022/1023)
p−PLCG1(Y783)
p−PDGFRB(Y1009)
p−STAT6(Y641)
p−SYK(Y525/526)
p−EGFR(Y1068)
p−FAK(Y576/577)
p−PYK2(Y402)
p−ERBB2(Y1221/1222)
p−FGFR1(Y653/654)
p−STAT1(Y701)
p−EGFR(Y845)
p−PLCG2(Y759)
p−SRC(Y416)
p−MET(Y1234/1235)
p−EGFR(Y1173)
p−EGFR(Y1086)
0 1 2-1-2
b)
a)
0 0.2 0.4 0.6 0.8 1 1.2 1.4
Media
EGF(200ng/mL)
EGF(200ng/mL)+Aprotinin(1ug/mL)
EGF(200ng/mL)+GM6001(25uM)
EGF(200ng/mL)+TAPI−1(100uM)
45

0(0m)X4
EGF200(5m)X4
EGF200(15m)X4
EGF200CAPE(0m)X4
EGF200CAPE(5m)X4
EGF200CAPE(15m)X4
EGF200EGCG(0m)X4
EGF200EGCG(5m)X4
EGF200EGCG(15m)X4
EGF200N−Acetylcysteine(0m)X4
p−AKT(S473)
p−PKCPAN/G(T514)
p−P90RSK(S380)
p−FOXO1(S319)/…
p−STAT5(Y694)
p−ERBB4(Y1284)
p−KIT(C−)(Y719)
p−GAB1(Y627)
p−GSK3A(S21)
p−MEK(1/2)(S217/221)
p−PTEN(S380)
p−EGFR(Y845)
p−JAK1(Y1022/1023)
p−SHC(Y239/240)
p−ERK(T202/Y204)
p−FGFR1(Y653/654)
p−CREB(S133)
p−CRKII(Y221)
p−ERBB2(Y1221/1222)
p−PKCD(T505)
p−MET(Y1349)
p−MTOR(S2448)
p−SYK(Y525/526)
p−FOXO3A(S253)
p−ABL(C−)(Y245)
p−IRS1(S1101)
p−CDC2(Y15)
p−P70S6K(T421/S424)
p−STAT3(Y705)
p−4EBP1(S65)
p−GAB2(S159)
p−RAF(C−)(S259)
p−RAF(C−)(S338)
p−PDK1(S241)
p−CDK2(T160)
p−SRC(Y527)
p−CRKL(Y207)
p−SRC(Y416)
p−4EBP1(T37/46)
p−PI3KP85(Y458)P55(Y199)
p−IGF1RB (Y1135/1136) /…
p−PDGFRA(Y754)
p−STAT3(S727)
p−ZAP70(Y319)/SYK(Y352)
p−PLCG1(Y783)
p−FAK(Y576/577)
p−PLCG2(Y759)
p−MET(Y1234/1235)
p−STAT1(Y701)
p−STAT6(Y641)
p−EGFR(Y1068)
p−EGFR(Y1086)
p−PDGFRB(Y1009)
p−EGFR(Y1173)
p−PYK2(Y402)
p−SHP2(Y542)
0 1 2-1-2
0 0.5 1 1.5
Media
EGF(200ng/mL)
EGF(200ng/mL)+CAPE(10uM)
EGF(200ng/mL)+EGCG(100uM)
EGF(200ng/mL)+N−Acetylcysteine(30mM)
a)
b)
Figure 11: a) The clustergram of phosphorylation kinetics is shown for Panel 4 of small molecules. b) Relative cell
46

0(0m)X5
EGF200(5m)X5
EGF200(15m)X5
EGF200DPI(0m)X5
EGF200DPI(5m)X5
EGF200DPI(15m)X5
EGF200ClostridiumToxinB(0m)X5
EGF200PHPS2(0m)X5
EGF200PHPS2(5m)X5
EGF200PHPS2(15m)X5
p−FOXO3A(S253)
p−MTOR(S2448)
p−RAF(C−)(S338)
p−STAT3(S727)
p−SHC(Y239/240)
p−RAF(C−)(S259)
p−GAB2(S159)
p−FOXO1(S319)/…
p−CDK2(T160)
p−PKCPAN/G(T514)
p−IRS1(S1101)
p−PDK1(S241)
p−P70S6K(T421/S424)
p−PKCD(T505)
p−P90RSK(S380)
p−AKT(S473)
p−4EBP1(T37/46)
p−CREB(S133)
p−SRC(Y527)
p−PTEN(S380)
p−ABL(C−)(Y245)
p−GSK3A(S21)
p−CDC2(Y15)
p−CRKL(Y207)
p−4EBP1(S65)
p−MEK(1/2)(S217/221)
p−PI3KP85(Y458)P55(Y199)
p−IGF1RB (Y1135/1136) /…
p−CRKII(Y221)
p−ERBB4(Y1284)
p−PDGFRA(Y754)
p−GAB1(Y627)
p−ERK(T202/Y204)
p−PDGFRB(Y1009)
p−MET(Y1349)
p−ZAP70(Y319)/SYK(Y352)
p−STAT3(Y705)
p−JAK1(Y1022/1023)
p−ERBB2(Y1221/1222)
p−PLCG1(Y783)
p−PYK2(Y402)
p−EGFR(Y845)
p−STAT5(Y694)
p−MET(Y1234/1235)
p−KIT(C−)(Y719)
p−EGFR(Y1173)
p−EGFR(Y1068)
p−SYK(Y525/526)
p−FAK(Y576/577)
p−SRC(Y416)
p−FGFR1(Y653/654)
p−PLCG2(Y759)
p−STAT6(Y641)
p−SHP2(Y542)
p−STAT1(Y701)
p−EGFR(Y1086)
0 1 2-1-2
0 0.5 1 1.5
Media
EGF(200ng/mL)
EGF(200ng/mL)+DPI(25uM)
EGF(200ng/mL)+Clostr. Toxin B(2ng/mL)
EGF(200ng/mL)+PHPS1(20uM)
a)
b)
47

EGF200(0m)X6
EGF200(5m)X6
EGF200(15m)X6
EGF200DPI(0m)X6
EGF200DPI(5m)X6
EGF200DPI(15m)X6
EGF200PP2(0m)X6
EGF200PP2(5m)X6
EGF200PP2(15m)X6
EGF200EMBPTP1BI(0m)X6
p−4EBP1(T37/46)
p−PTEN(S380)
p−FOXO1(S319)/…
p−SRC(Y527)
p−CDK2(T160)
p−4EBP1(S65)
p−CDC2(Y15)
p−ABL(C−)(Y245)
p−RAF(C−)(S338)
p−STAT3(S727)
p−GAB2(S159)
p−PDK1(S241)
p−RAF(C−)(S259)
p−AKT(S473)
p−CRKL(Y207)
p−CREB(S133)
p−PI3KP85(Y458)P55(Y199)
p−PDGFRA(Y754)
p−PKCD(T505)
p−FOXO3A(S253)
p−MTOR(S2448)
p−CRKII(Y221)
p−SRC(Y416)
p−P70S6K(T421/S424)
p−PKCPAN/G(T514)
p−GSK3A(S21)
p−MET(Y1349)
p−P90RSK(S380)
p−IGF1RB (Y1135/1136) /…
p−IRS1(S1101)
p−SHC(Y239/240)
p−PYK2(Y402)
p−STAT5(Y694)
p−GAB1(Y627)
p−MEK(1/2)(S217/221)
p−ERK(T202/Y204)
p−SHP2(Y542)
p−EGFR(Y845)
p−ZAP70(Y319)/SYK(Y352)
p−FGFR1(Y653/654)
p−ERBB4(Y1284)
p−ERBB2(Y1221/1222)
p−PDGFRB(Y1009)
p−JAK1(Y1022/1023)
p−EGFR(Y1173)
p−STAT3(Y705)
p−FAK(Y576/577)
p−KIT(C−)(Y719)
p−STAT1(Y701)
p−SYK(Y525/526)
p−MET(Y1234/1235)
p−EGFR(Y1068)
p−PLCG1(Y783)
p−STAT6(Y641)
p−PLCG2(Y759)
p−EGFR(Y1086)
0 1 2-1-2
a)
b)
0 0.5 1 1.5
Media
EGF(200ng/mL)
EGF(200ng/mL)+DPI(10uM)
EGF(200ng/mL)+PP2(200nM)
EGF(200ng/mL)+EMBPTP1BI(25uM)
48

−150 −100 −50 0 50 100 150
1.4
1.6
1.8
2
2.2
2.4
2.6
2.8
3
p−CDK2(T160)[0m]
p−PI3KP85(Y458)[0m]P55(Y199)[0m]
p−PKCPAN/G(T514)[0m]
p−CDC2(Y15)[5m]
p−EGFR(Y1086)[5m]
p−EGFR(Y845)[5m]
p−ERBB4(Y1284)[5m]p−ERK(T202/Y204)[5m]
p−FGFR1(Y653/654)[5m]
p−GSK3A(S21)[5m]
p−JAK1(Y1022/1023)[5m]
p−MET(Y1234/1235)[5m]
p−MTOR(S2448)[5m]
p−PLCG2(Y759)[5m]
p−PTEN(S380)[5m]
p−STAT3(Y705)[5m]
p−4EBP1(T37/46)[15m]
p−CDK2(T160)[15m]
p−CRKL(Y207)[15m]
p−EGFR(Y1086)[15m]
p−EGFR(Y1173)[15m]
p−ERBB4(Y1284)[15m]
p−MEK(1/2)[15m](S217/221)[15m]
p−P38MAPK(T180/Y182)[15m]
p−P90RSK(S380)[15m]
p−PYK2(Y402)[15m]
p−SAPK/JNK(T183/Y185)[15m]
Percent inhibition of Aprotinin on EGF stimulation of specified phosphometric
−log10(p−value)
Figure 14: Volcano plots for each of inhibitor combination. The x-axis reﬂects the percent inhibition of each phosphome-
tric given the addition of the chemical perturbant. The y-axis reﬂects the scaled p-value.
49

−60 −40 −20 0 20 40 60 80
1.4
1.6
1.8
2
2.2
2.4
2.6
2.8
3
3.2
3.4
p−EGFR(Y1086)[0m]
p−ERK(T202/Y204)[0m]
p−MET(Y1234/1235)[0m]p−P38MAPK(T180/Y182)[0m]
p−PLCG1(Y783)[0m]
p−PLCG2(Y759)[0m]
p−PTEN(S380)[0m]
p−STAT1(Y701)[0m]
p−ZAP70(Y319)[0m]/SYK(Y352)[0m]
p−ABL(C−)[5m](Y245)[5m]
p−CREB(S133)[5m]
p−ERBB4(Y1284)[5m]
p−ERK(T202/Y204)[5m]
p−FAK(Y576/577)[5m]
p−MTOR(S2448)[5m]
p−P38MAPK(T180/Y182)[5m]
p−P90RSK(S380)[5m]
p−PLCG2(Y759)[5m]
p−RAF(C−)[5m](S259)[5m]
p−RAF(C−)[5m](S338)[5m]
p−AKT(S473)[15m]
p−CDK2(T160)[15m]
p−CRKII(Y221)[15m]
p−ERK(T202/Y204)[15m]
p−FAK(Y576/577)[15m]
p−GAB2(S159)[15m]
p−IGF1RB (Y1135/1136)[15m] /p−InsR…
p−MTOR(S2448)[15m]
p−P38MAPK(T180/Y182)[15m]
p−PDGFRB(Y1009)[15m]
p−PLCG2(Y759)[15m]
p−RAF(C−)[15m](S259)[15m]
p−RAF(C−)[15m](S338)[15m]
Percent inhibition of CAPE on EGF stimulation of specified phosphometric
−log10(p−value)

−150 −100 −50 0 50 100 150
1.5
2
2.5
3
3.5
4
p−4EBP1(T37/46)[0m]
p−EGFR(Y1086)[0m]
p−P90RSK(S380)[0m]
p−STAT6(Y641)[0m]
p−ABL(C−)[5m](Y245)[5m]
p−FGFR1(Y653/654)[5m]
p−P38MAPK(T180/Y182)[5m]
p−PDGFRA(Y754)[5m]
p−RAF(C−)[5m](S259)[5m]
p−SHC(Y239/240)[5m]
p−SHP2(Y542)[5m]
p−STAT1(Y701)[5m]
p−EGFR(Y1086)[15m]
p−ERBB2(Y1221/1222)[15m]
p−ERK(T202/Y204)[15m]
p−FGFR1(Y653/654)[15m]
p−IGF1RB (Y1135/1136)[15m] /p−InsR…
p−MET(Y1234/1235)[15m]
p−MTOR(S2448)[15m]
p−STAT6(Y641)[15m]
p−SYK(Y525/526)[15m]
Percent inhibition of Clostridium Toxin B on specified phosphometric
−log10(p−value)

−150 −100 −50 0 50 100 150
1.5
2
2.5
3
3.5
p−RAF(C−)[0m](S338)[0m]
p−CREB(S133)[5m]
p−FOXO3A(S253)[5m]
p−GSK3A(S21)[5m]
p−MEK(1/2)[5m](S217/221)[5m]
p−PLCG2(Y759)[5m]
p−STAT3(S727)[5m]
p−STAT5(Y694)[5m]
p−ZAP70(Y319)[5m]/SYK(Y352)[5m]
p−4EBP1(T37/46)[15m]
p−CREB(S133)[15m]
p−CRKL(Y207)[15m]
p−EGFR(Y1086)[15m]
p−EGFR(Y845)[15m]
p−ERK(T202/Y204)[15m]
p−FAK(Y576/577)[15m]
p−FGFR1(Y653/654)[15m]
p−JAK1(Y1022/1023)[15m]
p−MEK(1/2)[15m](S217/221)[15m]
p−MET(Y1234/1235)[15m]
p−PDFGRA(Y849)[15m]/PDGFRB(Y857)[15m]
p−PI3KP85(Y458)[15m]P55(Y199)[15m]
p−PLCG2(Y759)[15m]
p−PYK2(Y402)[15m]
p−S6RIBPROT(S240/244)[15m]
p−STAT3(S727)[15m]
p−STAT6(Y641)[15m]
p−ZAP70(Y319)[15m]/SYK(Y352)[15m]
Percent inhibition of DPI(10 uM) on specified phosphometric
−log10(p−value)

−150 −100 −50 0 50 100 150
1.4
1.6
1.8
2
2.2
2.4
2.6
2.8
3
p−RAF(C−)[0m](S338)[0m]
p−STAT3(Y705)[0m]
p−ABL(C−)[5m](Y245)[5m]
p−CREB(S133)[5m]
p−CRKL(Y207)[5m]
p−ERK(T202/Y204)[5m]
p−FGFR1(Y653/654)[5m]
p−KIT(C−)[5m](Y719)[5m]
p−P38MAPK(T180/Y182)[5m]
p−P90RSK(S380)[5m]
p−SHP2(Y542)[5m]
p−STAT6(Y641)[5m]
p−ZAP70(Y319)[5m]/SYK(Y352)[5m]
p−4EBP1(T37/46)[15m]
p−EGFR(Y1173)[15m]
p−ERBB2(Y1221/1222)[15m]
p−ERBB4(Y1284)[15m]
p−ERK(T202/Y204)[15m]
p−IRS1(S1101)[15m]
p−P38MAPK(T180/Y182)[15m]
p−ZAP70(Y319)[15m]/SYK(Y352)[15m]
Percent inhibition of DPI(25 uM) on specified phosphometric
−log10(p−value)

−300 −200 −100 0 100 200 300 400 500 600 700
1.5
2
2.5
3
3.5
p−AKT(S473)[0m]
p−EGFR(Y1173)[0m]
p−ERBB4(Y1284)[0m]
p−ERK(T202/Y204)[0m]
p−GAB2(S159)[0m]
p−IGF1RB (Y1135/1136)[0m] /p−InsR…
p−P38MAPK(T180/Y182)[0m]
p−PDK1(S241)[0m]
p−EGFR(Y1068)[5m]
p−EGFR(Y1173)[5m]
p−ERBB4(Y1284)[5m]
p−ERK(T202/Y204)[5m]
p−FAK(Y576/577)[5m]
p−IGF1RB (Y1135/1136)[5m] /p−InsR…
p−JAK1(Y1022/1023)[5m]
p−MTOR(S2448)[5m]
p−P38MAPK(T180/Y182)[5m]
p−PDK1(S241)[5m]
p−PLCG2(Y759)[5m]
p−RAF(C−)[5m](S259)[5m]
p−RAF(C−)[5m](S338)[5m]
p−STAT1(Y701)[5m]
p−AKT(S473)[15m]
p−EGFR(Y1173)[15m]
p−ERBB4(Y1284)[15m]
p−GAB2(S159)[15m]
p−GSK3A(S21)[15m]
p−IGF1RB (Y1135/1136)[15m] /p−InsR…
p−MET(Y1234/1235)[15m]
p−P38MAPK(T180/Y182)[15m]
p−PYK2(Y402)[15m]
p−STAT1(Y701)[15m]
Percent inhibition of EGCG on EGF stimulation of specified phosphometric
−log10(p−value)

−150 −100 −50 0 50 100 150
1.4
1.6
1.8
2
2.2
2.4
2.6
2.8
3
3.2
3.4
p−CDK2(T160)[0m]
p−CRKII(Y221)[0m]
p−FAK(Y576/577)[0m]
p−FGFR1(Y653/654)[0m]
p−GAB2(S159)[0m]
p−MEK(1/2)[0m](S217/221)[0m]
p−MET(Y1234/1235)[0m]
p−SRC(Y416)[0m]
p−ABL(C−)[5m](Y245)[5m]
p−EGFR(Y1068)[5m]
p−EGFR(Y845)[5m]
p−ERK(T202/Y204)[5m]
p−FAK(Y576/577)[5m]
p−GSK3A(S21)[5m]
p−MEK(1/2)[5m](S217/221)[5m]
p−MET(Y1234/1235)[5m]
p−PI3KP85(Y458)[5m]P55(Y199)[5m]
p−PLCG1(Y783)[5m]
p−PLCG2(Y759)[5m]
p−RAF(C−)[5m](S338)[5m]
p−STAT1(Y701)[5m]
p−STAT5(Y694)[5m]
p−ZAP70(Y319)[5m]/SYK(Y352)[5m]
p−4EBP1(T37/46)[15m]
p−CREB(S133)[15m]
p−CRKL(Y207)[15m]
p−EGFR(Y1086)[15m] p−EGFR(Y845)[15m]
p−ERBB2(Y1221/1222)[15m]
p−FAK(Y576/577)[15m]
p−FGFR1(Y653/654)[15m]
p−GAB1(Y627)[15m]
p−GAB2(S159)[15m]
p−JAK1(Y1022/1023)[15m]
p−MET(Y1234/1235)[15m]
p−P38MAPK(T180/Y182)[15m]
p−P90RSK(S380)[15m]
p−PI3KP85(Y458)[15m]P55(Y199)[15m]
p−PLCG2(Y759)[15m]
p−PYK2(Y402)[15m]
p−SHC(Y239/240)[15m]
p−STAT1(Y701)[15m]
p−STAT6(Y641)[15m]
p−ZAP70(Y319)[15m]/SYK(Y352)[15m]
Percent inhibition of EMBPTP1BI on specified phosphometric
−log10(p−value)

−150 −100 −50 0 50 100 150
1.4
1.6
1.8
2
2.2
2.4
p−AKT(S473)[0m]
p−CRKII(Y221)[0m]
p−SHC(Y239/240)[0m]
p−STAT3(Y705)[0m]
p−ZAP70(Y319)[0m]/SYK(Y352)[0m]
p−CDK2(T160)[5m]
p−ERBB4(Y1284)[5m]
p−FGFR1(Y653/654)[5m]
p−MET(Y1234/1235)[5m]
p−PDK1(S241)[5m]
p−RAF(C−)[5m](S259)[5m]
p−RAF(C−)[5m](S338)[5m]
p−ABL(C−)[15m](Y245)[15m]
p−CDC2(Y15)[15m]
p−P38MAPK(T180/Y182)[15m]
Percent inhibition of GM6001 on EGF stimulation of specified phosphometric
−log10(p−value)

−50 0 50 100 150
1.4
1.6
1.8
2
2.2
2.4
2.6
2.8
3
p−AKT(S473)[0m]
p−EGFR(Y1068)[0m]
p−ERBB4(Y1284)[0m]
p−IGF1RB (Y1135/1136)[0m] /p−InsR…
p−MET(Y1234/1235)[0m]
p−PDK1(S241)[0m]
p−PYK2(Y402)[0m]
p−RAF(C−)[0m](S259)[0m]
p−ZAP70(Y319)[0m]/SYK(Y352)[0m]
p−ABL(C−)[5m](Y245)[5m]
p−ERBB4(Y1284)[5m] p−ERK(T202/Y204)[5m]
p−FAK(Y576/577)[5m]
p−P38MAPK(T180/Y182)[5m]
p−ERBB2(Y1221/1222)[15m]
p−ERK(T202/Y204)[15m]
p−FGFR1(Y653/654)[15m]
p−MEK(1/2)[15m](S217/221)[15m]
p−MET(Y1234/1235)[15m]
p−P38MAPK(T180/Y182)[15m]
p−PDK1(S241)[15m]
p−PKCD(T505)[15m]
p−PYK2(Y402)[15m]
p−RAF(C−)[15m](S259)
p−STAT1(Y701)[15m]
Percent inhibition of N−Ac on EGF stimulation of specified phosphometric
−log10(p−value)

−150 −100 −50 0 50 100 150
1.4
1.6
1.8
2
2.2
2.4
2.6
2.8
3
3.2
3.4
p−4EBP1(T37/46)[0m]
p−CREB(S133)[0m]
p−ERK(T202/Y204)[0m]
p−RAF(C−)[0m](S259)[0m]
p−STAT3(Y705)[0m]
p−ZAP70(Y319)[0m]/SYK(Y352)[0m]
p−CDC2(Y15)[5m]
p−CREB(S133)[5m]
p−EGFR(Y1086)[5m]
p−ERBB2(Y1221/1222)[5m]
p−ERK(T202/Y204)[5m]
p−GAB1(Y627)[5m]
p−GSK3A(S21)[5m]
p−MEK(1/2)[5m](S217/221)[5m]
p−P38MAPK(T180/Y182)[5m]
p−PYK2(Y402)[5m]
p−RAF(C−)[5m](S259)[5m]
p−RAF(C−)[5m](S338)[5m]
p−SHC(Y239/240)[5m]
p−STAT6(Y641)[5m]
p−ABL(C−)[15m](Y245)[15m]
p−CDC2(Y15)[15m]
p−EGFR(Y1086)[15m]
p−ERBB2(Y1221/1222)[15m]
p−MTOR(S2448)[15m]
p−P38MAPK(T180/Y182)[15m]
p−STAT1(Y701)[15m]
p−STAT6(Y641)[15m]
Percent inhibition of PHPS2 on specified phosphometric
−log10(p−value)

−150 −100 −50 0 50 100 150
1.5
2
2.5
3
3.5
4
4.5
p−4EBP1(S65)[5m]
p−4EBP1(T37/46)[5m]
p−AKT(S473)[5m]
p−CDK2(T160)[5m]
p−CRKL(Y207)[5m]
p−EGFR(Y1068)[5m]
p−EGFR(Y1086)[5m]
p−EGFR(Y1173)[5m]
p−EGFR(Y845)[5m]
p−ERK(T202/Y204)[5m]
p−GSK3A(S21)[5m]
p−JAK1(Y1022/1023)[5m]
p−MET(Y1234/1235)[5m]
p−P90RSK(S380)[5m]
p−PLCG2(Y759)[5m]
p−SHC(Y239/240)[5m]
p−SRC(Y527)[5m]
p−4EBP1(S65)[15m]
p−4EBP1(T37/46)[15m]
p−AKT(S473)[15m]
p−CDK2(T160)[15m]
p−CRKL(Y207)[15m]
p−EGFR(Y1068)[15m]
p−EGFR(Y1086)[15m]
p−EGFR(Y845)[15m]
p−GAB2(S159)[15m]
p−GSK3A(S21)[15m]
p−IGF1RB (Y1135/1136)[15m] /p−InsR…
p−MEK(1/2)[15m](S217/221)[15m]
p−P70S6K(T421/S424)[15m]
p−P90RSK(S380)[15m]
p−PLCG2(Y759)[15m]
p−PYK2(Y402)[15m]
p−RAF(C−)[15m](S259)[15m]
p−SHC(Y239/240)[15m]
p−STAT3(Y705)[15m]
p−STAT5(Y694)[15m]
p−STAT6(Y641)[15m]
p−ZAP70(Y319)[15m]/SYK(Y352)[15m]
Percent inhibition of PP2 on EGF stimulation of specified phosphometric
−log10(p−value)

−150 −100 −50 0 50 100 150
1.5
2
2.5
3
3.5
p−CDC2(Y15)[0m]
p−CDK2(T160)[0m]
p−EGFR(Y845)[0m]
p−FGFR1(Y653/654)[0m]
p−GAB2(S159)[0m]
p−GSK3A(S21)[0m]
p−PDK1(S241)[0m]
p−FAK(Y576/577)[5m]
p−PYK2(Y402)[5m]
p−RAF(C−)[5m](S259)[5m]
p−RAF(C−)[5m](S338)[5m]
p−ABL(C−)[15m](Y245)[15m]
p−CREB(S133)[15m]
p−CRKL(Y207)[15m]
p−EGFR(Y1086)[15m]
p−EGFR(Y845)[15m]
p−ERBB2(Y1221/1222)[15m]
p−ERK(T202/Y204)[15m]
p−FAK(Y576/577)[15m]
p−FGFR1(Y653/654)[15m]
p−GAB1(Y627)[15m]
p−IRS1(S1101)[15m]
p−JAK1(Y1022/1023)[15m]
p−MET(Y1234/1235)[15m]
p−PDK1(S241)[15m]
p−PI3KP85(Y458)[15m]P55(Y199)[15m]
p−PLCG1(Y783)[15m]
p−PLCG2(Y759)[15m]
p−PYK2(Y402)[15m]
p−RAF(C−)[15m](S259)[15m]
p−SHC(Y239/240)[15m]
p−SRC(Y416)[15m]
p−SRC(Y527)[15m]
p−STAT1(Y701)[15m]
p−STAT3(S727)[15m]
p−STAT6(Y641)[15m]
p−ZAP70(Y319)[15m]/SYK(Y352)[15m]
Percent inhibition of PP2(200 nM) on specified phosphometric
−log10(p−value)

−150 −100 −50 0 50 100 150
1.5
2
2.5
3
3.5
4
p−AKT(S473)[0m]
p−CRKL(Y207)[0m]
p−MEK(1/2)[0m](S217/221)[0m]
p−AKT(S473)[5m]
p−CDK2(T160)[5m]
p−CREB(S133)[5m]
p−EGFR(Y1068)[5m]
p−EGFR(Y1086)[5m]
p−EGFR(Y1173)[5m]
p−ERK(T202/Y204)[5m]
p−FAK(Y576/577)[5m]
p−GSK3A(S21)[5m]
p−MEK(1/2)[5m](S217/221)[5m]
p−P38MAPK(T180/Y182)[5m]
p−P90RSK(S380)[5m]
p−PDK1(S241)[5m]
p−PLCG2(Y759)[5m]
p−RAF(C−)[5m](S259)[5m]
p−MEK(1/2)[15m](S217/221)[15m]
p−MTOR(S2448)[15m]
p−STAT1(Y701)[15m]
Percent inhibition of TAPI1 on EGF stimulation of specified phosphometric
−log10(p−value)

−150 −100 −50 0 50 100 150
1.5
2
2.5
3
3.5
p−SRC(Y527)[0m]
p−4EBP1(S65)[5m]
p−4EBP1(T37/46)[5m]
p−AKT(S473)[5m]
p−CRKL(Y207)[5m]
p−EGFR(Y1068)[5m]
p−EGFR(Y1086)[5m]
p−EGFR(Y1173)[5m]
p−EGFR(Y845)[5m]
p−ERK(T202/Y204)[5m]
p−GSK3A(S21)[5m]
p−MEK(1/2)[5m](S217/221)[5m]
p−MET(Y1234/1235)[5m]
p−PLCG2(Y759)[5m]
p−PYK2(Y402)[5m]
p−4EBP1(T37/46)[15m]
p−AKT(S473)[15m]
p−CDK2(T160)[15m]
p−EGFR(Y1068)[15m]
p−EGFR(Y845)[15m]
p−ERBB2(Y1221/1222)[15m]
p−GAB2(S159)[15m]
p−GSK3A(S21)[15m]
p−IGF1RB (Y1135/1136)[15m] /p−InsR…
p−MEK(1/2)[15m](S217/221)[15m]
p−P90RSK(S380)[15m]
p−PLCG2(Y759)[15m]
p−PYK2(Y402)[15m]
p−RAF(C−)[15m](S259)[15m]
p−SHC(Y239/240)[15m]
p−SRC(Y527)[15m]
p−STAT3(Y705)[15m]
p−STAT5(Y694)[15m]
p−STAT6(Y641)[15m]
p−ZAP70(Y319)[15m]/SYK(Y352)[15m]
Percent inhibition of U73122 on EGF stimulation of specified phosphometric
−log10(p−value)

−150 −100 −50 0 50 100 150
1.5
2
2.5
3
3.5
p−KIT(C−)[0m](Y719)[0m]
p−P90RSK(S380)[0m]
p−4EBP1(S65)[5m]
p−4EBP1(T37/46)[5m]
p−AKT(S473)[5m]
p−CRKL(Y207)[5m]
p−EGFR(Y1068)[5m]
p−EGFR(Y1173)[5m]
p−EGFR(Y845)[5m]
p−ERBB2(Y1221/1222)[5m]
p−FOXO1(S319)[5m]/FOXO4(S262)[5m]
p−GAB2(S159)[5m]
p−GSK3A(S21)[5m]
p−MET(Y1234/1235)[5m]
p−PI3KP85(Y458)[5m]P55(Y199)[5m]
p−PLCG2(Y759)[5m]
p−PTEN(S380)[5m]
p−SHP2(Y542)[5m]
p−STAT1(Y701)[5m]
p−4EBP1(T37/46)[15m]
p−AKT(S473)[15m]
p−CDK2(T160)[15m]
p−CRKL(Y207)[15m]
p−EGFR(Y1068)[15m]
p−EGFR(Y1086)[15m]
p−EGFR(Y845)[15m]
p−ERBB2(Y1221/1222)[15m]
p−GAB2(S159)[15m]
p−GSK3A(S21)[15m]
p−MEK(1/2)[15m](S217/221)[15m]
p−PLCG2(Y759)[15m]
p−PYK2(Y402)[15m]
p−RAF(C−)[15m](S259)[15m]
p−SHC(Y239/240)[15m]
p−SRC(Y527)[15m]
p−STAT6(Y641)[15m]
p−ZAP70(Y319)[15m]/SYK(Y352)[15m]
Percent inhibition of Wortmannin on EGF stimulation of specified phosphometric
−log10(p−value)

Experimental vs. model: Simple linear regression
Viability(Experimental)Viability(Experimental)
Viability (Model) Viability (Model)
−2 −1 0 1 2
−2
−1
0
1
2
p−EGFR(Y1086)[5m]
n = 1
Q2
=0.717
R2
=0.745
−2 −1 0 1 2
−2
−1
0
1
2
p−STAT1(Y701)[5m]
n = 1
Q2
=0.687
R2
=0.725
−2 −1 0 1 2
−2
−1
0
1
2
p−GAB2(S159)[5m]
n = 1
Q2
=0.563
R2
=0.610
−2 −1 0 1 2
−2
−1
0
1
2
p−PDK1(S241)[15m]
n = 1
Q2
=−0.018
R2
=0.086
Viability(Experimental)Viability(Experimental)
Prediction fit to all data
Prediction fit without
modeled condition included
Line of unity corresponding
to perfect fit
Figure 15: Comparison of simple linear regression models. A comparison of simple linear regression models
reveals signiﬁcant predictors for explaining cell viability. The R2 and Q2
LOO metrics are given for each
optimized regression method to quantify its ﬁtness and predictive capacity, respectively. The number of
predictors used in each algorithm is depicted by n. Simple least squares regression was implemented using
four highly-correlated phosphoprotein signaling metrics. Solid green circles show predicted response for
conditions that are trained on the complete data set. Open blue circles show predictions that were trained on
data omitting the predicted condition.
64

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
False positive rate
Truepositiverate
Inference of 100 Node In Silico Network: 3
DIONESUS (AUC=.633)
Spearman Correlations (AUC=.610)
GENIE3 (AUC=.628)
Inferelator (AUC=.566)
TIGRESS (AUC=.610)
Random (AUC=.500)
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
False positive rate
Truepositiverate
DIONESUS (AUC=.585)
GENIE3 (AUC=.595)
TIGRESS (AUC=.524)
Random (AUC=.500)
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Recall
Precision
DIONESUS (AUC=.244)
GENIE3 (AUC=.208)
TIGRESS (AUC=.201)
Random (AUC=.117)
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Recall
Precision
DIONESUS (AUC=0.188)
Spearman Correlations (AUC=0.157)
GENIE3 (AUC=0.186)
Inferelator (AUC=0.159)
TIGRESS (AUC=0.159)
Random (AUC=0.134)
a. In Silico Network 2: ROC Curve b. In Silico Network 3: ROC Curve
c. In Silico Network 2: PR Curve d. In Silico Network 3: PR Curve
Figure 16: DIONESUS accurately reconstructs a signaling network from a kinetic model derived from two
one-hundred node in silico networks . (a and b) The receiving-operating characteristic (ROC) curves
for DIONESUS and top-performing inference algorithms for the reconstruction of two 100-node in silico
network from kinetic time-course data. Higher accuracy of inference corresponds to a higher Area Under
the ROC curve (AUROC). (c and d) The precision-recall (PR) curves corresponding to the same analyses
displayed in part d are shown. A higher area under the PR curve (AUPR) corresponds to a more accurate
algorithm.
65

P1
P2
P3
P1
P2
P3
P1
P2
P31.06
1.34
1.25
0.77
0.84
0.42
Gold Standard Network
DIONESUS-inferred Network
(All edges)
DIONESUS-inferred Network
(Thresholded at VIP = 1.00)
P1
P3
P2
P31.14
1.34
1.25
0.90
0.87
0.48
P3
P2
P30.90
0.83
1.32
0.85
0.81
0.90
In silico
Network 1
In silico
Network 2
In silico
Network 3
P3
P3
P1
P2
P1
P2
P1
P2P2
VIP score
P1P1
Figure 17: Three separate 3-node in silico toy models were constructed using GeneNetWeaver to demon-
strate the strengths and limitations of inferring the directionality and the activating or in-
hibitory nature of each inferred edge using DIONESUS. Each model was created from a linear cascade
motif of the structure: P1→P2→P3. While none of these networks were inferred with 100% accuracy, in
each case, the algorithm correctly inferred whether each real edge was activating or inhibitory. In addition,
the real edges (e.g. P1→P2) always reflected higher confidence (indicated by the VIP score) than the reverse,
false positive edge (e.g. P2→P1). Furthermore, the indirect edge from P1→P3 reflected the least confidence
and was consistently below the edge threshold in all three examples. Taken together, the DIONESUS
algorithm was able to uncover the most salient features of the in silico models.
66

Daughter Node
ParentNode
DIONESUS - GENIE3
10 20 30 40 50 60
10
20
30
40
50
60
ParentNode
Daughter Node
DIONESUS - Inferelator
10 20 30 40 50 60
10
20
30
40
50
60
ParentNode
Daughter Node
DIONESUS - TIGRESS
10 20 30 40 50 60
10
20
30
40
50
60
ParentNode
Daughter Node
DIONESUS - Random
10 20 30 40 50 60
10
20
30
40
50
60
DIONESUS
DIONESUS
GENIE3
TIGRESS
Inferelator
Random
1.00
1.00
1.00
1.00
1.00
Jaccard similarity index
(Threshold = 500 edges)
DIONESUS and comparison
algorithm do not detect edges
DIONESUS and comparison
algorithm both detect edge
DIONESUS detects edge but
comparison algorithm does not
Comparison algorithm detects
edge but DIONESUS does not
A.
B.
COMPARISON OF ADJACENCY
MATRICES
GENIE3
TIGRESS
Inferelator
Random
0.51 0.36 0.54
0.34 0.34
0.34
0.08
0.08
0.08
0.08
Figure 18: DIONESUS performs similarly to top-performing network inference algorithms with a fraction
of the computational expense. a) The adjacency matrix between all 60 phosphosites was calculated for
the DIONESUS, GENIE3, Inferelator, and TIGRESS algorithms using default parameters. Each algorithm
was thresholded at 500 edges to provide a sufﬁcient edge count for comparison. As a negative control, a
single random matrix was created by assigning 500 edges with equal probability to each position in the
matrix (excluding self-edges). A comparison of the overlap for each adjacency matrix is shown. b) The
Jaccard similarity index, a measure of adjacency matrix similarity, between two algorithms are listed in the
table. DIONESUS and TIGRESS shared the most edges in common suggesting similar preferences in edge
detection.
67

Figure 19: ImageJ software was used to quantify microwestern arrays where bands were difficult to differ-
entiate by visual examination. In cases where there were multiple bands in close proximity in a lane
of the microwestern array, the intensity of pixels from the top to the bottom of the microwestern lane was
assessed using the ImageJ software. A line was manually drawn from the top to the bottom of the lane and
the ’Plot Intensity’ tool was used to create a plot of the pixel intensity across the length of the line. This
figure reflects the microwestern array lane of A431 cell treated with EGF+INS stimulation and blotted
with the p-c-MET(Y1349) antibody. The value for each band is the integral of the pixel intensities between
inflection points on each side of a peak. This method removes human variability in the quantification of
band intensities.
68

DIONESUS algorithm reveals new drug targets from dynamic phosphoproteomic networks

Recomendados

Recomendados

Mais conteúdo relacionado

Mais procurados

Mais procurados (20)

Destaque

Destaque (6)

Semelhante a DIONESUS algorithm reveals new drug targets from dynamic phosphoproteomic networks

Semelhante a DIONESUS algorithm reveals new drug targets from dynamic phosphoproteomic networks (20)

Mais de 1901 Atos das Policias Internacionais

Mais de 1901 Atos das Policias Internacionais (20)

Último

Último (11)

DIONESUS algorithm reveals new drug targets from dynamic phosphoproteomic networks