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Supplementary Materials for
Using Functional Signature Ontology (FUSION) to Identify Mechanisms of Action for Natural Products
Malia B. Potts, Hyun Seok Kim, Kurt W. Fisher, Youcai Hu, Yazmin P. Carrasco, Gamze
Betul Bulut, Yi-Hung Ou, Mireya L. Herrera-Herrera, Federico Cubillos, Saurabh Mendiratta, Guanghua Xiao, Matan Hofree, Trey Ideker, Yang Xie, Lily Jun-shen Huang,
Robert E. Lewis,* John B. MacMillan,* Michael A. White*
*Corresponding author. E-mail: [email protected] (M.A.W.); [email protected] (J.B.M.); [email protected] (R.E.L.)
Published 15 October 2013, Sci. Signal. 6, ra90 (2013)
DOI: 10.1126/scisignal.2004657 This PDF file includes:
Materials and Methods: Natural Products and Producing Organisms Fig. S1. Characteristics of the reporter genes used for FUSION mapping. Fig. S2. Mapping functional similarity among miRNAs. Fig. S3. Mapping functional similarity among kinases and kinase accessory proteins. Fig. S4. Using FUSION mapping to identify miRNAs and kinases that regulate autophagy. Fig. S5. Representative flow cytometry data and characterization of the role of BMP2K in autophagy and erythrocyte development. Fig. S6. SRMS as an inhibitor of autophagy. Fig. S7. Using FUSION mapping to identify natural product inhibitors of autophagy and migration. Fig. S8. Using FUSION mapping to identify inhibitors of AKT signaling. Fig. S9. Natural product structure determination. Fig. S10. Profiles of reporter gene expression changes in response to each of 1124 genetic perturbations. Fig. S11. Analysis of correlation among reporter gene expression and between distance metrics. Fig. S12. Estimation of FDR from P value density distribution, rank sum as an example. Legends for tables S1 to S5
Other Supplementary Material for this manuscript includes the following: (available at www.sciencesignaling.org/cgi/content/full/6/297/ra90/DC1)
Table S1 (Microsoft Excel format). Reporter gene expression in response to individual genetic and chemical perturbations in HCT116 cells. Table S2 (Microsoft Excel format). Endogenous miRNA expression. Table S3 (Microsoft Excel format). MiRNA-siRNA oligonucleotide seed identities. Table S4 (Microsoft Excel format). Kinase inhibition screen (KinaseProfiler). Table S5 (Microsoft Excel format). Maintenance and transfection conditions for cell lines used in this study.
Materials and Methods: Natural Products and Producing Organisms
Isolation of natural product fractions
Natural product extracts from marine invertebrates were generated in the following way; ~ 1 g of
freeze-dried tissue was dissected into small pieces and soaked in a 2:1 mixture of
methanol/dichloromethane for 24 hours, filtered to remove animal material, and concentrated in
vacuo to give a complex crude mixture. The crude extract was then sequentially extracted with n-
hexane, ethyl acetate, n-butanol, and H2O to give four fractions for biological screening.
The library of microbial and invertebrate natural product fractions were subjected to liquid
chromatography-mass spectrometry (LC-MS) analysis using an Agilent Model 6130 single
quadrupole mass spectrometer with a HP1200 HPLC. A photodiode array detector provided a
chemical fingerprint of all fractions on the basis of molecular weight and UV profile. Fractions
were dereplicated using various compound databases, including Antibase, Reaxys, and Scifinder
Scholar.
Producing organisms
Standard procedures for 16S rRNA analysis were used for phylogenetic characterization of
bacterial strains (36). Bacterial strain SNB-003 was derived from a sediment sample collected
from Trinity Bay, Galveston, TX (29° 42.419’N, 94° 49165’W). . Based on 16S rRNA analysis
the strain was determined to be Salinispora arenicola. Bacterial strain SNA-048 was derived
from a sediment sample collected in Galveston Bay, TX and determined to be 98% similar to
Bacillus hunanensis. Bacterial strain SNB-019 was derived from a sediment sample collected in
Galveston Bay, TX and determined to be a Streptomyces bacillaris based on 16S rRNA analysis.
Table 1 provides a summary of compounds derived from these species and reported in this study.
Purification of natural products from SNB-019
Bacterial strain SNB-019 was isolated as previously described (37). 16S rRNA analysis
identified the strain as Streptomyces bacillaris. Five bioactive compounds were purified by
chromatography (fig. S9 A-H). The SNB-019 extract (36 mg) was fractionated by Sephadex LH-
20 (25 g, 1.5 65 cm, eluted with MeOH), followed by reversed phase HPLC (Phenomenex
Luna, Phenyl-Hexyl, 250 × 10.0 mm, 2.5 mL/min, 5 μm, UV = 210 nm) using a gradient solvent
system from 60% to 80% CH3CN (0.01% Formic acid) over 30 min to afford SNB-019-cmp1
(bafilomycins D, 1), SNB-019-cmp2 (bafilomycin A1, 2) SNB-019-cmp3 (bafilomycin B1, 3),
SNB-019-cmp4 (bafilomycin B2, 4) and a non bafilomycin natural product SNB-019-cmp5
(5)(37). The bafilomycins D, A1, B1, and B2 were assigned based on comparison of the UV,
NMR, and MS data with those of reference compounds. The assignment of NMR signals for
bafilomycin D and B1 were further corroborated with COSY, HSQC, and HMBC data.
Bafilomycin D (1): white solid; UV (MeCN) λmax 245, 285 nm; 1H NMR (fig. S9A, 600 MHz,
CDCl3) and 13
C NMR (125 MHz, CD3Cl3) are consistent with previously reported NMR
data(38); ESI-MS [M + Cl - H]- m/z 639.3, [M + Na]
+ m/z 627.3; HRESIMS [ [M - H]
- m/z
603.3908 (C35H55O8, calcd 603.3902).
Bafilomycin A1 (2): yellow solid; UV (MeCN) λmax 249, 285 nm; 1H NMR (fig. S9C, 600 MHz,
CDCl3) and 13
C NMR (125 MHz, CD3Cl3) are consistent with previously reported NMR
data(39); ESI-MS [M + Cl - H]- m/z 657.4, [M + Na]
+ m/z 645.4; HRESIMS [M + Na]
+ m/z
645.3987 (C35H58O9Na, calcd 645.3979).
Bafilomycin B1 (3): yellow solid; λmax 249, 290, 345 nm; 1H NMR (fig. S9E, 600 MHz, CDCl3)
and 13
C NMR (125 MHz, CD3Cl3) are consistent with previously reported NMR data(40); ESI-
MS [M - H]- m/z 814.4; HRESIMS [M + Na]
+ m/z 838.4368 (C44H65NO13Na, calcd 838.4354).
Bafilomycin B2 (4): white solid; UV (MeCN) λmax 249, 288, 340 nm; ESI-MS [M - H]- m/z
828.4, [M + Na]+ m/z 852.3;
1H NMR (fig. S9G, 600 MHz, CDCl3) are consistent with
previously reported NMR data(40); HRESIMS [M + Na]+ m/z 852.4521 (C45H67NO13Na, calcd
852.4510) and [M - H]- m/z 828.4553 (C45H66NO13, calcd 828.4540).
Purification of natural products from SNB-003
Strain SNB-003 was isolated from a sediment sample collected in Trinity Bay, Galveston, TX.
Desiccated sediment sample was stamped on a seawater based media (10 g starch, 4 g yeast
extract, 2 g peptone, 15 g agar, 1 L sH2O) containing rifampicin (50 mg/L) and cycloheximide
(10 mg/L). Phylogentic analysis using 16S rRNA analysis revealed the strain to be identical to S.
arenicola. The dried crude extract from 10 L of S. arenicola (7.2 g) was purified using solvent
partition (MeOH) , the methanol soluble portion (4.6g) was further partitioned using EtOAc and
H2O (1:1 mixture). The ethyl acetate layer (1.3g) was purified via reversed phase flash column
chromatography, eluting with a step gradient of H2O and MeOH (90:10-100:0) collecting 13
fractions. Fraction 11 (SNB-003-int3, 120mg) was purified by reversed phase HPLC (
Phenomenex Luna, C18, 250×21.2 mm, 8.0 ml/min, 10µm, UV=254nm) using a gradient solvent
system from 35% to 70% MeOH (0.1% formic acid) over 35 min, collecting 7 fractions. Fraction
3 (SNB-003-int4, 9.7mg) was further purified by reversed phase HPLC (Phenomenex Luna, C18,
250×10 mm, 2.5 ml/min, 5µm, UV=254nm), a gradient solvent system was utilized (60% to
100% MeOH + 0.1% formic acid over 28 min) to give SNB-003-cmp1, which has not been
structurally characterized.
SNB-003-cmp1: λmax (log ε) 243 (4.4), 267 (4.4), 292 (4.8), 322 (4.4), 335 (4.2), 356 (4.0), 372
(4.0). ESI-MS m/z 467.2 [M + H]+, 511.2 [M+ Formic acid-H]
-. HRESIMS m/z 467.2082.
1H
NMR (fig. S9N).
Purification of natural products from SNA-048
Fraction SNA-048-7, which clustered with the siRNA for DDR2, was further purified via
sequential flash ODS chromatography (step gradient of MeOH/H2O), Sephadex LH-20 (MeOH),
and reversed phase HPLC (Phenomenex Luna, Phenyl-Hexyl, 250 x 10 mm) using CH3CN:H2O,
gave SNA-048-7-2, subsequently named discoipyrrole A (Fig. 5F, Table 1).
Discoipyrrole A (6) yellow solid; [α]D 0 (c 1.2 MeOH); UV (MeOH) λmax (log ε) 214 (4.25), 263
(3.82), 334 (3.29), 396 (3.46); ESI-MS m/z 464.2 [M + Na]+, 440.2 [M – H]
-. HRESIMS m/z
442.1652 [M + H]+
(C27H24NO5, calcd 442.1655), 440.1550 [M – H]- (C27H22NO5, calcd
440.1498). 1
H and 13
C NMR data (fig. S9I,J).
Structural assignment of discoipyrrole A was accomplished by conversion to the bis(p-
bromobenzoyl) derivative.
bis(p-bromobenzoyl) discoipyrrole A. A solution of discoipyrrole A (4.0 mg) in dry CH2Cl2
(2.0 mL) was treated with triethylamine (4.0 µL) under nitrogen. A solution of p-bromo-benzoyl
chloride (6.0 mg) in dry CH2Cl2 (300 µL) was added at 0 oC. After completion of the reaction
(about 2 h) as monitored by TLC, the solvent was removed under N2. The residue was purified
by normal phase HPLC (Phenomenex Luna, siliga, 250 × 10.0 mm, 2.5 mL/min, 5 mm, UV =
340 nm) using a gradient solvent system from 10% to 40% EtOAC in hexane over 20 min to
afford a yellow powder of bis(p-bromobenzoyl) discoipyrrole A (4.7 mg, tR = 15.8 min) which
was crystallized from MeOH/CHCl3 (10:1) to give yellow crystals. HRESIMS m/z 806.0381 [M
+ H]+
(C41H30Br2NO7, calcd 806.0389). The X-ray crystal structure has been deposited as CCDC
#910866 (fig. S9M).
N
O O
O
HO
HO
N
O O
O
O
O
O
Br
O
Br
discoipyrrole A bis(p-BrBz)-discoipyrrole A
pBrBzCl
Et3N, CH2Cl2
Fig. S1. Characteristics of the reporter genes used for FUSION mapping. (A) Selection of
endogenous reporter genes. Expression variation is shown for six high variance reporter
selections (red; ACSL5, BNIP3L, LOXL2, ALDOC, NDRG1, BNIP3) and two low variance
selections (blue; HPRT and PPIB). The top indicates the expression of these genes across 3554
unique drug instances in MCF7 cells as reported in the Connectivity Map (CMAP)
(http://www.broadinstitute.org/cmap/) (6). The bottom indicates the expression of these genes
across 86 non small cell lung cancer lines (http://www.bcgsc.ca/project/bomge/coexpression
(13). (B) Chemical and genetic perturbations were assayed for effects on the expression of six
endogenous reporter genes in HCT116 cells. Perturbations were performed separately in
triplicate. Correlations among the reporter signatures imply functional similarity. (C) Normalized
effect of the 426 miRNA mimics on the indicated transcript when individually introduced into
HCT116 cells. Expression values were normalized to two control genes (HPRT and PPIB),
averaged across triplicates, median normalized, and plotted as log2 values. Box represents 25th
-
75th
quartile with bar representing the median. Whiskers extend to 1.5 × the interquartile
distance, and outliers are indicated by circles. (D) Normalized expression of individual reporter
genes (y-axis) for the miRNA library as a function of the abundance of the corresponding
endogenous microRNA (x-axis). The y-axis is artificially truncated at -3 for ALDOC and
LOXL2. miRNAs with endogenous expression < 5,000 arbitrary units caused significantly more
variation in ACSL5 than miRNAs with endogenous expression > 5,000 arbitrary units (P < 0.01
by F test to compare variances). The variation in BNIP3L, ALDOC, LOXL2, BNIP3, and
NDRG1 expression was not significantly different between these groups (P > 0.10 by F test to
compare variances).
Sup. Figure 2
453768-3p.1220 221
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ALD
OC
N
DR
G1
BN
IP3L
B
NIP
3 A
CS
L5
LO
XL2
hsa-miR-192 hsa-miR-7 hsa-miR-124a hsa-miR-7 hsa-miR-299-3p hsa-miR-185 hsa-miR-512-5p hsa-miR-28 hsa-miR-491 hsa-miR-363* hsa-miR-95 hsa-miR-378 hsa-miR-379 hsa-miR-517c hsa-miR-452 hsa-miR-138 hsa-miR-138 hsa-miR-147 hsa-miR-511 hsa-miR-494 hsa-miR-511 hsa-miR-134 hsa-miR-29b hsa-miR-29a hsa-miR-29b hsa-miR-29c hsa-miR-215 hsa-miR-182* hsa-miR-7 hsa-miR-512-5p hsa-miR-194 hsa-miR-497 hsa-miR-383 hsa-miR-432* hsa-miR-380-5p hsa-miR-510 hsa-miR-193b hsa-miR-100 hsa-miR-296 hsa-miR-24 hsa-miR-324-5p hsa-miR-483 hsa-miR-489 hsa-miR-148a hsa-miR-152 hsa-miR-423 hsa-miR-485-5p hsa-miR-99b hsa-miR-328 hsa-miR-148b hsa-miR-24 hsa-miR-765 hsa-miR-17-3p hsa-miR-151 hsa-let-7a hsa-let-7b hsa-let-7a hsa-let-7c hsa-let-7f hsa-let-7f hsa-let-7g hsa-let-7a hsa-miR-320 hsa-miR-369-5p hsa-miR-488 hsa-miR-103 hsa-miR-103 hsa-miR-107 hsa-miR-516-5p hsa-miR-516-5p hsa-miR-187 hsa-miR-425 hsa-miR-542-5p hsa-miR-142-3p hsa-miR-126 hsa-miR-196b hsa-miR-200a* hsa-miR-203 hsa-miR-124a hsa-miR-526a hsa-miR-526a hsa-miR-19b hsa-miR-19a hsa-miR-19b hsa-miR-34a hsa-miR-449 hsa-miR-127 hsa-miR-520a* hsa-miR-9* hsa-miR-9* hsa-miR-9* hsa-miR-519e* hsa-miR-220 hsa-miR-453 hsa-miR-768-3p hsa-miR-768-3p hsa-miR-518b hsa-miR-10a hsa-miR-98 hsa-miR-10b hsa-miR-376a hsa-miR-202 hsa-miR-125b hsa-miR-125a hsa-miR-490 hsa-miR-125b hsa-miR-433 hsa-miR-452* hsa-miR-197 hsa-miR-122a hsa-miR-331 hsa-miR-515-3p hsa-miR-515-3p hsa-miR-154 hsa-miR-362 hsa-miR-324-3p hsa-miR-496 hsa-miR-517a hsa-miR-522 hsa-miR-329 hsa-miR-137 hsa-miR-135a hsa-miR-99a hsa-miR-484 hsa-miR-135a hsa-miR-135b hsa-miR-329 hsa-miR-516-3p hsa-miR-516-3p hsa-miR-516-3p hsa-miR-516-3p hsa-miR-767-3p hsa-miR-487b hsa-miR-22 hsa-miR-337 hsa-miR-527 hsa-miR-527 hsa-miR-544 hsa-miR-15a hsa-miR-508 hsa-miR-27a hsa-miR-27b hsa-miR-128a hsa-miR-486 hsa-miR-30a-5p hsa-miR-30c hsa-miR-30c hsa-miR-30d hsa-miR-149 hsa-miR-30b hsa-miR-31 hsa-miR-345 hsa-miR-367 hsa-miR-370 hsa-miR-32 hsa-miR-513 hsa-miR-363 hsa-miR-513 hsa-miR-144 hsa-miR-25 hsa-miR-222 hsa-miR-377 hsa-miR-129 hsa-miR-129 hsa-miR-384 hsa-miR-132 hsa-miR-199a hsa-miR-485-3p hsa-miR-155 hsa-miR-524* hsa-miR-376a* hsa-miR-199a hsa-miR-150 hsa-miR-199b hsa-miR-770-5p hsa-miR-181a hsa-miR-181c hsa-miR-181a hsa-miR-181b hsa-miR-181b hsa-miR-181d hsa-miR-198 hsa-miR-325 hsa-miR-518f hsa-miR-518a hsa-miR-454-5p hsa-miR-16 hsa-miR-16 hsa-miR-15b hsa-miR-195 hsa-miR-365 hsa-let-7d hsa-miR-519e hsa-let-7i hsa-miR-340 hsa-miR-493 hsa-miR-509 hsa-miR-219 hsa-miR-219 hsa-miR-20a hsa-miR-448 hsa-miR-21 hsa-miR-139 hsa-miR-769-5p hsa-miR-503 hsa-miR-769-5p hsa-miR-23a hsa-miR-526b hsa-miR-223 hsa-miR-23b hsa-miR-495 hsa-miR-92 hsa-miR-92 hsa-miR-451 siCONTROL hsa-miR-191 hsa-miR-302c* hsa-miR-213 hsa-miR-136 siCONTROL hsa-miR-323 siCONTROL hsa-miR-143 hsa-miR-515-5p hsa-miR-515-5p hsa-miR-412 hsa-miR-518a hsa-miR-365 hsa-miR-205 siCONTROL siCONTROL hsa-miR-202* hsa-miR-520d* siCONTROL hsa-miR-302a* hsa-miR-211 hsa-miR-376a hsa-miR-146b hsa-miR-146a hsa-miR-368 hsa-miR-380-3p hsa-miR-498 hsa-miR-18a hsa-miR-18b hsa-miR-335 hsa-miR-539 hsa-miR-410 hsa-miR-106b hsa-miR-191* hsa-miR-518a-2* hsa-miR-514 hsa-miR-492 hsa-miR-521 hsa-miR-521 hsa-miR-196a hsa-miR-196a hsa-miR-346 hsa-miR-154* hsa-miR-526c hsa-miR-526a hsa-miR-501 hsa-miR-376b hsa-miR-526c hsa-miR-526c hsa-miR-526c hsa-miR-526c hsa-miR-768-5p hsa-miR-487 hsa-miR-526c hsa-miR-424 hsa-miR-507 hsa-miR-141 hsa-miR-140 hsa-miR-200a hsa-miR-767-5p hsa-miR-802 hsa-miR-96 hsa-miR-373 hsa-miR-765 hsa-miR-93 hsa-miR-520d hsa-miR-372 hsa-miR-302b hsa-miR-302a hsa-miR-302d hsa-miR-520g hsa-miR-520e hsa-miR-520a hsa-miR-526b* hsa-miR-520b hsa-miR-520c hsa-miR-519c hsa-miR-519a hsa-miR-519a hsa-miR-342 hsa-miR-523 hsa-miR-208 hsa-miR-30e-3p hsa-miR-101 hsa-miR-186 hsa-miR-153 hsa-miR-153 siKSR siKSR hsa-miR-199a* hsa-miR-199a* hsa-miR-128b hsa-miR-361 hsa-miR-189 hsa-miR-339 hsa-miR-26a hsa-miR-330 hsa-miR-371 hsa-miR-101 hsa-miR-200b hsa-miR-200c hsa-miR-26b hsa-miR-26a hsa-miR-382 hsa-miR-429 hsa-miR-194 hsa-miR-767-5p hsa-miR-299-5p hsa-miR-802 hsa-miR-105 hsa-miR-105 hsa-miR-409-5p hsa-miR-499 hsa-miR-30e-5p hsa-miR-190 hsa-miR-505 hsa-miR-18a* hsa-miR-204 hsa-miR-545 hsa-miR-338 hsa-miR-514 hsa-miR-188 hsa-miR-500 hsa-miR-374 hsa-miR-409-3p hsa-miR-30a-3p hsa-miR-33 hsa-miR-218 hsa-miR-218 hsa-miR-126* hsa-miR-455 hsa-miR-106a hsa-miR-20b hsa-miR-518c* hsa-miR-519d hsa-miR-450 hsa-miR-17-5p siKSR siKSR siKSR siKSR siKSR siKSR siKSR siKSR hsa-miR-758 hsa-miR-758 hsa-miR-210 hsa-miR-221 hsa-miR-518e hsa-miR-518c hsa-miR-520h hsa-miR-518d hsa-miR-326 hsa-miR-502 hsa-miR-373* hsa-miR-801 hsa-miR-375 hsa-miR-801 hsa-miR-302b* hsa-miR-514 hsa-miR-542-3p hsa-miR-184 hsa-miR-224 hsa-miR-133a hsa-miR-133b hsa-miR-432 hsa-miR-193a hsa-miR-34b hsa-miR-518f* hsa-miR-526a hsa-miR-768-5p hsa-miR-133a hsa-miR-517b hsa-miR-450 hsa-miR-431 hsa-miR-525 hsa-miR-506 hsa-miR-216 hsa-miR-145 hsa-miR-1 hsa-miR-206 hsa-miR-770-5p hsa-miR-183 hsa-miR-668 hsa-miR-182 hsa-miR-519b hsa-miR-512-3p hsa-miR-512-3p hsa-miR-520f hsa-miR-9 hsa-miR-9 hsa-miR-9 hsa-miR-422b hsa-miR-422a hsa-miR-769-3p hsa-miR-769-3p hsa-let-7e hsa-miR-142-5p hsa-miR-493-3p hsa-miR-668 hsa-miR-214 hsa-miR-671 hsa-miR-671 hsa-miR-1 hsa-miR-212 hsa-miR-217 hsa-miR-302c hsa-miR-517* hsa-miR-517* hsa-miR-381 hsa-miR-517* siCONTROL hsa-miR-34c hsa-miR-369-3p siCONTROL siCONTROL siCONTROL hsa-miR-454-5p hsa-miR-130a hsa-miR-130b hsa-miR-766 hsa-miR-124a hsa-miR-301 hsa-miR-454-3p hsa-miR-454-3p hsa-miR-767-3p hsa-miR-525* hsa-miR-524 hsa-miR-766 hsa-miR-504
−4 −2 0 2 4
Expression (log2)
Fig. S2. Mapping functional similarity among miRNAs. Left: Reporter gene expression shown
as a clustered heat map. Right: miRNA mimics plotted as a neighboring-joining tree. See table
S1 for details.
Sup. Figure 3
DGKB
C9ORF12
ALS2CR2
HAK
FRAP1
MGC45428
MAP3K8
PRKCA
DAPK2
STK19
CHEK1
GRK6
HSMDPKIN
MAPK8IP2
AKAP1
RAC1
MAP3K10
CSNK1D
AKAP4
IRAK1
MERTK
ACVR1CLYK5
DKFZP586B1621MPZL1EPHA1
KISCD4
STYK1
DUSP22FLJ10761
DYRK3SIK2MASTL
GK
MAPK8IP1
GUCY2CMAP2K5
EPHB2
TBK1
TGFBR2
HK2
STK22B
KALRN
ACVRL1
ERN1
EDN2
TJP2
RAPGEF3BTK
PTK7RPS6KA5
CDKL1
STK38L
PDPK1STK10
ERK8
CLK3
HIPK3
BRAF
MYLK2
DKFZP761P0423
ANGPT4NPR2
PAPSS2
NTRK1ABL1 STK36
CKB AK3L1
COL4A3BP
SCYL1
AK5NEK1
NME1
PANK1
DGKG
DMPK
DUSP7
PDGFRA
PDGFRB
GRK4
PRKAA2PRKG1
PDK3
NRBPPRPSAP1
PXKCAMK4
RPS6KA6
FES
THNSL1
NEK2
PPP4C
AKAP8
PRKACG
PHKG1MYO3A
TEK
MGC4796
DUSP1
BCKDK
TTBK2
ITGB1BP1
TRIM
CDC2
PRKDC
PRKCH
GALK1
PRKCN
CSNK1E
AZU1
GMFB
PIK4CB
PRKAR1A
APEG1
PFKFB1WIF1
FLJ12476
RYK
SGK2
ARAF1
SMG1
STK32C
RNASEL
SNF1LK
MATK
STK32BMVD
ERBB4
PPP2CA
RPS6KA4
RAGE
ROCK2
TK1
DLG4
MUSK
YWHAQ
PMVK
NME6CDKL2PRKWNK1
DUSP4
ACVR1B
ROR1
LOC115704
MIDORI
ACVR2B
STK25
MPP3
MAPKAPK2
DUSP6
PGK2
EXOSC10
DDR2
XYLB
AKAP7
RASGRF2
CHKA
NEK3
CDKN1B
AATK
TXK
PPP1R1B
PAK3
CSNK1A1
MAP3K3
CDKN2B
STK17B
IKBKAP
DGKQ
LIMK2
ULK2
TAO1.1KIAA1765
CDKN2C
RPS6KA1
PIP5K2B
ADRBK2
STK33
JAK1
DUSP10
SQSTM1
PRKRA
CDC2L1
TLK2
TSKS
CSNK2B
PAK6
PCTK2
EPHA3
NLK
SRC
PRKCSH
STK29
RP2
CKMT1
PIP5KL1
C7ORF16
NEK8
CSF1R
TYRO3
SYK
PSKH2
PITPNM3 FRDA
EPHA8
INSRR
TTN
VRK2
PRKCQ
DNAJC3
EPHA7
LAK
ADCK1
PRKACA
TAO1.2
ALK
TLR3
CSNK1G3
FLJ13052
DCAMKL1
LOC149420
DLG2
MAP3K5
FYN
PRKWNK3
STK17A
CLK2
UMP-CMPK
MAP4K5
STK22C
INSR
ROS1
ULK1
BMP2K
YWHAH
MAPK14
PRPS1L1
C6ORF199
GCKMYO3B
MAPK13
ACVR2
MBIP
CAMK2D
SMAD7GK2
MAP4K3
RAPGEF4
MAP2K4
CHEK2
MKNK1
PANK3
MVK
CASK
PGK1
PRKCI
CDC7
SNRK
KIAA1361
PRKACB
CKMT2
IKBKE
CDKN1A
PRKAG1
CDC42BPB
ITPKBDYRK1A
MAPK10
STK32A
NEK4
PRKY
MAP3K6
ATR
PCTK1
PRKCL2
ADKLMTK2
PDK1CERK GRK1 HRI
PAGPAK7
MAPK7
EIF2AK3
JIK
PRKX
ACK1
PHKA2
CLK4
CXCL10
AGTR2
DAPK3
FASTK
ILKAPJAK2
CARKL
DKFZp434C1418
TYK2
CINP
DGKE
DGUOK
RPS6KB1
CAMK1
PTPRR
IRAK2
CSNK1G1
DCK
AK7
AK1
HUNK
AURKC
EPHA4
NTRK2PDK4
MAPK9MAP3K12
GRK7
FYBAKAP13MST1R
PACE-1LIMK1
BMPR1ASTK16
MAP3K2
CDK5
BRDT
MAPK6
MAKTRPM6
EPHA5TXNDC3
BMPR2
CDKL3PRKG2
VRK3
P15RSPRKWNK2
EGFRMAST2
LATS1
MAP3K14PAK2
RAF1
PTK6
MKNK2
PFKFB4
CARD10 SEPHS2
ITK
OSR1 KIF13BLATS2
EKI1
NRG3MGC16169
VRK1PYCS
MGC42105
EPHB1
GMFG
KIAA1639
AXLMARK4
NTRK3
PI4KII
NEK7
MAPK1MAP4K4 IHPK1
MAP2K7
KHKPTPRG
DLG1
CDK8PHKG2
PTK9L
PDXKCAMK2B
MAP2K1IP1
CDC42BPA
SGKL
DYRK4 BCR
EPHA2
NYD-SP25PDK2
MAPK8
KIAA1804
CSKPSKH1GSG2
PCK1
PRKCDPLK2
PRKAA1SLKPFKLPKIA
PRKD2
AVPR1A
STK4STK24
CCL4ABI1
PIK3R3FN3KRP
MAP2K2ATM
CDKN1CPIK3CASTK23
DKFZP434C131MAP3K7
PRPS2DGKAKIAA1811 MPP1
NBEA
MET CDK5R1
PFKP
ASPMAPK4ADRA1A
MAPKAPK5
MAP3K1MELK
CSNK2A1CHKB
PFTK1 CSNK1G2CRK7GUCY2D RPS6KA3
CLK1
CCRK
TAF1L
EPHB4
PTPRT
KIAA0999
TRIB1
MYLK
NME5
TEC
HK3
NME2
TEX14
BDKRB2
PTK9
CDK4PPP2CB
PASK
HK1
GRK5AKAP6
CDKN3
GSK3A
PINK1
CIT
HSPB8
CKS1B
FLJ10842
TLK1HIPK1
DAPK1
SRMS
NAGK
LRRK1
CDC2L5ZAP70
CAMKK2
TLR1
PRKCB1
EEF2K
AK2
RPS6KB2
UCK1DTYMK
AKAP5
ADRBK1
AKT2
CDK7
AKT3ALS2CR7
CSNK2A2
DDR1
CNKSR1
WEE1
AKT1
CDK10
CDK9
ZAK
PACSIN1
TNIK
SPHK1
GALK2
TIE
PRKWNK4 ILK
PHKA1
TAF1
PIM2
PANK4
TPK1
BMX
PTK2B
FGR
MRC2
PCTK3
CDK11
PRKCABP
ITPKA
PRKCL1
RIPK3
TRIB2
TLR4
BUB1
CDK3
PKN3
NEK6
PIK3CG
DYRK2
BRD2
TTBK1
HIPK2
PIM1
JAK3
DUSP5
MAPKAPK3
CKS2
SOCS1TP53RK
KSR2
FLJ10074
STK11
PRKAR2A
RIPK2
FLJ25006
TGFBR1
FLJ35107
PRKCM
TNNI3K
GUK1
PRKCE
IHPK2LMTK3
LOC340371
CDK5R2CAMK1G
C14ORF20
MAP3K4ERBB2
MARK3ADCK4
IL2
LCKARK5
TRIB3
STK6
MAP2K6
ITPK1
DGKZ
MGC5601
BLK
NME4
STK38
GAK
PLK4
SAST
STK39
RPS6KC1
KIAA1399
TK2
RPS6KA2
SPA17
DUSP8
CDK2
CAMK2G
ERBB3
MAP3K9
KDR
PRKCG
FGFR3
PRKR
CDKL5
ICK
GTF2H1
GSK3B
PTPRJ
MAPK3
PKMYT1
ROCK1
PIK3C2G
AMHR2
NPR1
PTK2
RFP
SPEC2
CDC2L2
NME3
PNKP
NEK9
TRIO
PTPN5TESK2
RFK
FLJ20574
CDK5RAP1
MAPK11
RBKS
HCK
STK22D
TRPM7
PCK2
CD3E
DYRK1B
ASK
RIPK1ROR2
BUB1B
LTK
SPHK2
ANKRD3
CHRM1
COASY
MAP2K1
CAMK2A
PRPF4B
URKL1
GAP43
MGC8407
MAPK12
TNK1
MARK2
AK3
SSTKADRA1B
EVI1
AKAP3
ANKK1
LCP2
IRAK3
AURKB
STK35
FER
PIP5K2A
PAK1
FLT1
ADCK5
FGFR1
PIK4CA
PKM2
PIK3R1
RIOK3
SHC1
BLNK
RIOK1
STK3
IRS1
MAP4K2
AAK1
C9ORF96
PIP5K2C
MAPK8IP3
PIK3C2B
IMPK
SRPK1
ADAM9
IKBKB
FRK
CDADC1
SOCS5
EPHB3
PIK3R4
NME7
MAP4K1
FN3K
FLJ23074
ABL2
EIF2AK4
SRPK2
SGKGUCY2F
MAP3K11
SNARK
CD7PRKAG3
MALT1
ACVR1
PRPS1
PIK3CBBMPR1B
KIT
MAP3K13
PKLR
RPS6KL1
MINKCARD14
CSNK1A1L
CRKL
PAPSS1
LOC91807MOS
STK31
IGF1RPI4K2B
TOPK
DLG3PFKM
PLK3
MPP2
PIK3R2
DGKD
HIPK4
ITPKC
TESK1
GNEFGFR4
YES1
ADCK2
MGC26597
NEK11
CKM
SCAP1
PAK4
UGP2
MST4
MAGI-3
C9ORF98
PFKFB2
DOK1TTK
CCL2
GFRA2TLR6
CDKN2DCALM3
COPB2
APPLCAMKK1
ADRB2
FLJ32685
FLT4 MAST3MARK1 AKAP11
LIMCAMK1DIHPK3
CDK6
PRKAR2B
TNFRSF10A FLJ34389UMPK
FLT3
DGKI
DUSP2
PFKFB3
PKIB
PIP5K1A
FUK
CDK5RAP3
AVPR1B
PRKCZPRPSAP2
LYNMAP2K3
SEPHS1
FLJ23356
FGFR2
PIK3C2A
RETEPHB6
CHUK
Fig. S3. Mapping functional similarity among kinases and kinase accessory proteins. A
neighbor joining tree of the Euclidean distance among reporter gene signatures for all siRNAs.
See table S1 for details.
Sup. Figure 4
A
ALD
OC
N
DR
G1
BN
IP3L
B
NIP
3 A
CS
L5
LO
XL2
KIAA1765/DCLK3 ULK2 LIMK2 TAOK2 ROS1 BMP2K ULK1 hsa-miR-410 SIK2 SQSTM1 hsa-miR-149 MAP3K3 hsa-miR-30a-5p hsa-miR-30c hsa-miR-30c hsa-miR-30d MAP2K5 hsa-miR-768-3p
ALD
OC
N
DR
G1
BN
IP3L
B
NIP
3 A
CS
L5
LO
XL2
FRAP1/mTOR MGC45428/DCLK2 EPHA3 NLK NEK8 DAPK1 SRMS CIT CKS1B HSPB8 NAGK hsa-miR-424 HIPK1 hsa-miR-33 FLJ10842/AGK TLK1 CDKN3 hsa-miR-507
-3.0
-2
.0
-1.0
0
.0
1.0
2
.0
3.0
Expression (log2)
C D
B afilo m yc in A1 T im co u rse
0 5 10 15 200.0
0.5
1.0
1.5
2.0
T ime (hours)G
FP-L
C3
Leve
l (Lo
g2)
E
S tarva tio n d ecreases L C 3-G F P leve ls
Control
EBSS
EBSS+BafA1-1.0
-0.5
0.0
0.5
Treatment
GFP
-LC
3 Le
vel (
Log2
)
F
ALD
OC
N
DR
G1
BN
IP3L
B
NIP
3 A
CS
L5
LO
XL2
CDC2 PRKDC AKAP11 LIM DUSP2 DGKI PFKFB3 PKIB hsa-miR-183 NBEA ACVRL1 ERN1 RAPGEF3 EDN2 TJP2 HK2 FRK hsa-miR-206 hsa-miR-1 AAK1 COPB2 AVPR1B PRKCZ CDK5RAP3 hsa-miR-130a hsa-miR-130b hsa-miR-766 EPHB6 PIK3C2A CDK6 FES CAMK1D IHPK3 MAST3 MARK1 PHKG1 GMFB PIK4CB PRKAR1A hsa-miR-9* hsa-miR-9* CHUK CCL2 FGFR2 PRPSAP2 LYN PANK4 ADRB2 FLJ32685 hsa-miR-488 PRKAR2B TNFRSF10A FUK PIP5K1A FLJ23356 RET hsa-miR-493-3p MAP2K3 FLT3 hsa-miR-671 SEPHS1 hsa-miR-214 hsa-miR-671 CALM3 C9ORF98 CAMKK1 APPL GFRA2 TLR6 hsa-miR-769-3p MAGI-3 hsa-miR-483 CDKN2D hsa-miR-454-3p PTK2B BMX hsa-miR-301 hsa-miR-454-3p RIPK3 PFKFB2 hsa-miR-767-3p hsa-miR-124a CD7 MAP3K11 SNARK DOK1 hsa-miR-151 TTK hsa-miR-769-3p PRKAG3 hsa-miR-103 hsa-miR-103 hsa-miR-107 FGFR3 PRKR KDR EPHB3 PIK3R4 PAK1 PIP5K2A FER PKM2 BLNK RIOK1 PIK3R1 RIOK3 SHC1 hsa-miR-34a STK3 MAP4K1 COASY PIK3C2B C9ORF96 hsa-miR-15a PIP5K2C hsa-miR-508 FGR CDK11 MRC2 PCTK3 PRKCABP hsa-miR-519b BMPR1B PIK3CB KIT hsa-miR-296 PRPS1 hsa-miR-9 hsa-miR-9 hsa-miR-9 hsa-miR-422b ADCK5 FGFR1 PIK4CA hsa-miR-182 hsa-miR-422a BLK FLT1 hsa-miR-512-3p hsa-miR-512-3p hsa-miR-520f FLJ34389 UMPK CDADC1 hsa-miR-381 SOCS5 ADAM9 hsa-miR-668 MGC5601 siCONTROL GAK siCONTROL siCONTROL STK38 PLK4 siCONTROLmiR siCONTROLmiR siCONTROLmiR NME4 hsa-miR-34c hsa-miR-369-3p hsa-miR-517* DGKZ hsa-miR-212 hsa-miR-217 hsa-miR-1 hsa-miR-302c hsa-miR-517* hsa-miR-517* IKBKB hsa-miR-668 DGKQ hsa-miR-22 CSNK1A1 CD4 DUSP22 DYRK3 hsa-miR-361 FLJ10761 STYK1 hsa-miR-511 PSKH2 DKFZP586B1621 MPZL1 hsa-miR-140 C7ORF16 ACVR1C hsa-miR-767-5p CSF1R TYRO3 SYK KALRN STK22B hsa-miR-802 AATK CDKN1B CHKA NEK3 FRAP1 MGC45428 EPHA3 NLK NEK8 DAPK1 SRMS CIT CKS1B HSPB8 NAGK hsa-miR-424 HIPK1 hsa-miR-33 FLJ10842 TLK1 CDKN3 hsa-miR-507 FASTK NME2 hsa-miR-210 PAK7 MVK MAPK7 GRK5 LRRK1 AKAP6 siKSR CCRK PTPRT KHK KIAA0999 siCONTROL GSK3A hsa-miR-518c hsa-miR-520h CDC42BPB ITPKB CDKN1A PRKAG1 siKSR DYRK1A STK32A MAPK10 siKSR hsa-miR-17-5p siKSR siKSR EPHB4 siKSR siKSR hsa-miR-106a MAP4K4 siKSR siKSR hsa-miR-20b IHPK1 MAP2K7 hsa-miR-519d PIK3C2G hsa-miR-20a hsa-miR-155 siKSR MGC42105 siKSR siKSR PYCS siKSR siKSR siKSR siKSR PRKG1 PDK3 hsa-miR-30a-3p CHEK2 MAP2K4 siKSRmiR siKSRmiR siKSRmiR siKSRmiR siKSRmiR VRK1 PTK6 hsa-miR-758 siKSR hsa-miR-758 PRKY PINK1 HK1 hsa-miR-526a hsa-miR-518f* siKSR siKSR hsa-miR-450 siKSR TXK hsa-miR-768-5p CERK LMTK2 PDK1 GK2 RAPGEF4 SMAD7 MKNK2 PFKFB4 EIF2AK3 JIK MGC16169 NRG3 EKI1 hsa-miR-18a* CSNK1G3 hsa-miR-505 SEPHS2 hsa-miR-190 hsa-miR-30e-5p ADK HUNK AURKC EPHA4 CKMT2 ATR NEK4 hsa-miR-518c* MAP3K6 DDR2 MAPKAPK2 MPP3 DUSP6 EXOSC10 PGK2 hsa-miR-496 hsa-miR-105 CDC2L5 IKBKE hsa-miR-514 ZAP70 RASGRF2 AKAP7 PPP1R1B NME5 HK3 TEC hsa-miR-502 hsa-miR-518d EPHB1 KIF13B LATS2 hsa-miR-514 ITK OSR1 hsa-miR-105 PDGFRA hsa-miR-499 AGTR2 GRK4 CAMKK2 PRKCB1 CLK4 PRKX ACK1 hsa-miR-200c PHKA2 PCTK1 PRKCL2 hsa-miR-101 hsa-miR-200b KIAA1361 CXCL10 PRKCI hsa-miR-218 CDC7 hsa-miR-218 SNRK NRBP hsa-miR-126* hsa-miR-455 ADCK1 EPHA7 TAO1 hsa-miR-330 TLR3 hsa-miR-545 hsa-miR-26a hsa-miR-26b PRKACA hsa-miR-429 hsa-miR-26a hsa-miR-382 hsa-miR-767-5p DLG2 DCAMKL1 LOC149420 ALK LAK hsa-miR-128b ACVR2 MYO3B MAPK13 MBIP MAPK9 MAP3K12 CASK PGK1 MKNK1 PANK3 CAMK2D MAP4K3 hsa-miR-524* hsa-miR-376a* GRK1 FLJ13052 KIS EPHA1 hsa-miR-199a PAG HRI hsa-miR-132 PRKCQ hsa-miR-485-3p hsa-miR-129 hsa-miR-129 hsa-miR-384 GCK hsa-miR-144 VRK2 hsa-miR-25 hsa-miR-222 hsa-miR-377 CDK10 CDC42BPA PDXK hsa-miR-99b MAP2K1IP1 CAMK2B hsa-miR-542-3p GMFG KIAA1639 AXL MARK4 P15RS EGFR MAST2 PRKWNK2 BRDT MAPK6 NPR2 PRKACB hsa-miR-204 hsa-miR-338 CDKN2B CDKL3 PRKG2 BMPR2 PTPRG EPHA5 VRK3 CLK2 UMP-CMPK hsa-miR-335 PRPS1L1 YWHAH MAPK14 TXNDC3 hsa-miR-31 siKSR siKSR MAK TRPM6 hsa-miR-221 NEK7 MAPK1 NTRK3 PI4KII hsa-miR-518e EPHA2 BCR DYRK4 SGKL NYD-SP25 hsa-miR-302b* PTK9L PDK2 MAPK8 MAP3K7 PRPS2 RPS6KA3 CLK1 KIAA1804 hsa-miR-518a-2* hsa-miR-514 LOC115704 ATM CDKN1C hsa-miR-801 hsa-miR-373* hsa-miR-326 TP53RK KSR2 CDK8 PHKG2 hsa-miR-106b hsa-miR-191* TNIK SPHK1 hsa-miR-521 hsa-miR-521 GK MAPK8IP1 GUCY2C PLK1 hsa-miR-539 PDPK1 CDKN2C RPS6KA1 PIP5K2B HIPK3 hsa-miR-146a BRAF MYLK2 hsa-miR-368 ADRBK2 JAK1 PAK3 hsa-miR-18a hsa-miR-18b STK33 hsa-miR-498 CDK9 HAK ALS2CR2 C9ORF12 MASTL PAPSS2 IKBKAP PRKAR2A AK3L1 CKB ABL1 STK36 hsa-miR-302a* IRAK1 MERTK hsa-miR-211 CLK3 STK25 MIDORI hsa-miR-376a hsa-miR-146b NTRK1 CAMK4 PRPSAP1 RPS6KA6 hsa-miR-371 PRKAA2 PXK hsa-miR-517a HCK STK22D PACSIN1 ZAK ERK8 PLK1 FLJ10074 STK11 hsa-miR-768-3p PRKRA CSNK2B PAK6 PCTK2 TSKS PRKWNK3 STK17A hsa-miR-380-3p TLK2 hsa-miR-526b LYK5 hsa-miR-189 hsa-miR-339 KIAA1765 ULK2 LIMK2 TAO1 ROS1 BMP2K ULK1 hsa-miR-410 SIK2 SQSTM1 hsa-miR-149 MAP3K3 hsa-miR-30a-5p hsa-miR-30c hsa-miR-30c hsa-miR-30d MAP2K5 hsa-miR-768-3p CHEK1 hsa-miR-10a hsa-miR-10b DAPK2 STK19 hsa-miR-376a DTYMK hsa-miR-331 ROR1 ACVR1B hsa-miR-515-3p hsa-miR-515-3p DLG4 YWHAQ PRKWNK1 hsa-miR-324-3p AKT1 hsa-miR-362 NME6 STK17B PLK1 hsa-miR-522 MUSK ANGPT4 hsa-miR-452* XYLB PMVK DKFZP761P0423 hsa-miR-433 hsa-miR-125b hsa-miR-125a hsa-miR-490 GRK6 AKAP3 ADRA1B EVI1 MARK2 MGC8407 MAPK12 CRK7 CSNK2A1 MELK MAPKAPK5 MAP3K1 GUCY2D hsa-miR-493 KIAA1811 MPP1 hsa-miR-340 DGKA hsa-miR-509 CRKL CARD14 hsa-miR-125b DUSP4 hsa-miR-98 TNK1 SGK2 hsa-miR-202 GAP43 APEG1 hsa-miR-519e* IGF1R TOPK PI4K2B PFKFB1 WIF1 hsa-miR-423 CSNK1G2 hsa-miR-100 MAP3K13 LOC91807 PAPSS1 STK31 MOS hsa-miR-17-3p AKAP8 PRKACG THNSL1 NEK2 PPP4C hsa-miR-9* RPS6KL1 hsa-miR-516-5p hsa-miR-516-5p hsa-miR-516-3p hsa-miR-516-3p ADCK4 ERBB2 MARK3 DYRK2 HIPK2 JAK3 PIM1 BRD2 hsa-miR-519a MINK ADRA1A hsa-miR-187 hsa-miR-425 MST1R PACE-1 hsa-miR-337 FYB AKAP13 LIMK1 MAP3K2 hsa-miR-542-5p hsa-miR-527 hsa-miR-527 ARAF1 hsa-miR-220 hsa-miR-544 CDK5R2 siCONTROL MAPKAPK3 CKS2 SOCS1 siCONTROL siCONTROL LOC340371 hsa-miR-373 IL2 PDK4 GRK7 hsa-miR-198 siCONTROL PKMYT1 RIPK2 C14ORF20 MAP3K4 hsa-miR-518f GNE FGFR4 YES1 TESK1 hsa-miR-454-5p hsa-miR-92 hsa-miR-451 siCONTROL siCONTROL siCONTROL CAMK1G hsa-miR-345 siCONTROL DUSP10 NTRK2 hsa-miR-513 hsa-miR-370 hsa-miR-32 FLJ35107 PRKCM LCK PRKCE ARK5 FRDA DNAJC3 EPHA8 RP2 STK29 SRC hsa-miR-21 PRKCSH hsa-miR-769-5p hsa-miR-139 FYN MAP3K5 hsa-miR-503 IHPK2 GUK1 FLJ25006 TGFBR1 hsa-miR-769-5p TNNI3K hsa-miR-202* hsa-miR-520d* ROCK1 hsa-miR-448 siCONTROL siCONTROL siCONTROL PCK2 MAP2K1 siCONTROL siCONTROL siCONTROL STK39 RPS6KC1 siCONTROL SAST siCONTROLmiR hsa-miR-765 DUSP5 hsa-miR-19b hsa-miR-19a hsa-miR-19b TTBK1 hsa-miR-96 siCONTROL PRKWNK4 GALK2 TIE HIPK4 PIM2 PHKA1 TAF1 ILK LCP2 MAPK8IP3 hsa-miR-518a DGKD MPP2 hsa-miR-518a CAMK2A PRPF4B URKL1 NME7 FN3K IMPK EPHB2 SRPK1 FLT4 hsa-let-7e hsa-miR-142-5p DLG3 PLK3 ITPKC PFKM BUB1B hsa-let-7i ACVR1 MALT1 hsa-let-7g PKLR hsa-let-7a hsa-let-7b hsa-let-7a hsa-let-7c hsa-let-7f hsa-let-7f hsa-let-7a hsa-miR-320 hsa-miR-369-5p BTK DGKB PITPNM3 CDC2L1 hsa-miR-200a hsa-miR-141 CDKL1 STK10 STK38L PTK7 RPS6KA5 TBK1 hsa-miR-412 TGFBR2 hsa-miR-515-5p ABL2 AMHR2 NPR1 siCONTROL RBKS siCONTROL MAPK11 hsa-miR-526c CD3E DYRK1B hsa-let-7d LTK hsa-miR-519e FLJ23074 hsa-miR-487 AURKB STK35 hsa-miR-213 GUCY2F ANKK1 EIF2AK4 SRPK2 SGK hsa-miR-136 siCONTROLmiR hsa-miR-323 siCONTROLmiR MST4 UGP2 PLK1 hsa-miR-223 hsa-miR-23b hsa-miR-495 PAK4 hsa-miR-143 hsa-miR-515-5p hsa-miR-191 hsa-miR-302c* MAP4K2 IRS1 hsa-miR-219 hsa-miR-219 LMTK3 CKMT1 PIP5KL1 hsa-miR-365 ROR2 hsa-miR-205 siCONTROLmiR siCONTROLmiR ASK RIPK1 hsa-miR-16 hsa-miR-16 hsa-miR-15b hsa-miR-195 hsa-miR-365 CDKL2 STK32B MATK SNF1LK ACVR2B MVD ERBB4 FLJ12476 SMG1 RYK hsa-miR-376b PPP2CA RPS6KA4 hsa-miR-329 RAGE STK32C RNASEL TAF1L hsa-miR-453 CSNK1D AKAP4 hsa-miR-196a hsa-miR-346 MAP3K8 PRKCA hsa-miR-154* AK3 SSTK ANKRD3 SPHK2 hsa-miR-501 hsa-miR-526a IRAK3 PTPN5 TESK2 CHRM1 CDK5RAP1 hsa-miR-526c RFK TRPM7 hsa-miR-492 hsa-miR-449 FLJ20574 hsa-miR-526c hsa-miR-526c hsa-miR-526c hsa-miR-526c hsa-miR-768-5p AKAP1 HSMDPKIN MAPK8IP2 RAC1 NEK9 TRIO hsa-miR-526a hsa-miR-526a MAP3K10 hsa-miR-142-3p hsa-miR-196a hsa-miR-196b hsa-miR-126 hsa-miR-200a* hsa-miR-203 ADCK2 NEK11 hsa-miR-23a MGC26597 CDK5R1 hsa-miR-128a ASP MAPK4 DLG1 PFKP hsa-miR-486 MET hsa-miR-27a CKM SCAP1 hsa-miR-27b CDK5 hsa-miR-325 STK16 hsa-miR-363 hsa-miR-513 hsa-miR-367 CDK3 NEK6 hsa-miR-520a* PRKCL1 PKN3 PIK3CG hsa-miR-127 TPK1 hsa-miR-525 DKFZP434C131 PIK3CA STK23 hsa-miR-93 hsa-miR-520d hsa-miR-372 hsa-miR-302b hsa-miR-302a hsa-miR-302d hsa-miR-520g hsa-miR-520e hsa-miR-520a hsa-miR-526b* hsa-miR-520b hsa-miR-520c hsa-miR-519c hsa-miR-519a hsa-miR-518b hsa-miR-342 hsa-miR-523 ERBB3 MAP3K9 CAMK2G ICK PLK1 BMPR1A PIK3R2 PLK1 hsa-miR-30b CDKL5 DUSP8 RPS6KA2 SPA17 CDK2 KIAA1399 TK2 hsa-miR-92 hsa-miR-454-5p TRIB3 GSK3B PTPRJ GTF2H1 PRKCG ITPK1 INSRR TTN MAPK3 hsa-miR-181a hsa-miR-181c MAP2K6 hsa-miR-181a hsa-miR-181b hsa-miR-181b hsa-miR-181d hsa-miR-525* hsa-miR-524 INSR MAP4K5 STK22C hsa-miR-150 hsa-miR-199a hsa-miR-216 hsa-miR-145 STK6 hsa-miR-199b hsa-miR-770-5p hsa-miR-770-5p CDK4 PASK PPP2CB BDKRB2 PLK1 PRKAA1 TRIB1 PTK9 MYLK STK24 AVPR1A STK4 hsa-miR-224 PFKL SPEC2 CSK PSKH1 PLK2 SLK PKIA PRKCD PCK1 hsa-miR-432 CCL4 hsa-miR-148b PIK3R3 ABI1 hsa-miR-328 hsa-miR-148a hsa-miR-152 FN3KRP MAP2K2 hsa-miR-801 hsa-miR-375 PNKP CDC2L2 NME3 PLK1 TEX14 hsa-miR-133a hsa-miR-133b RFP PTK2 hsa-miR-34b hsa-miR-124a TK1 ROCK2 hsa-miR-154 hsa-miR-197 PRKD2 hsa-miR-28 hsa-miR-133a hsa-miR-491 hsa-miR-517b hsa-miR-184 CSNK1E AZU1 CSNK1A1L hsa-miR-135b ITGB1BP1 TRIM DUSP1 BCKDK MYO3A TEK MGC4796 hsa-miR-99a hsa-miR-484 TTBK2 hsa-miR-135a hsa-miR-135a hsa-miR-137 BUB1 TRIB2 TLR4 GSG2 hsa-miR-122a hsa-miR-329 hsa-miR-516-3p hsa-miR-516-3p hsa-miR-767-3p ITPKA hsa-miR-506 PRKCH hsa-miR-487b CHKB PFTK1 hsa-miR-489 hsa-miR-24 hsa-miR-324-5p hsa-miR-485-5p GALK1 PRKCN hsa-miR-766 hsa-miR-24 hsa-miR-765 CSNK2A2 CSNK1G1 PLK1 PLK1 PLK1 PDGFRB DAPK3 hsa-miR-194 DCK hsa-miR-802 PLK1 hsa-miR-299-5p DGKE CINP hsa-miR-374 hsa-miR-409-3p C6ORF199 hsa-miR-500 hsa-miR-134 CDK7 DDR1 ADRBK1 AKT2 hsa-miR-29b hsa-miR-29a hsa-miR-29b hsa-miR-29c CNKSR1 WEE1 hsa-miR-194 IRAK2 hsa-miR-182* AK1 hsa-miR-512-5p AKT3 ALS2CR7 hsa-miR-7 hsa-miR-215 AKAP5 hsa-miR-497 EEF2K hsa-miR-193a hsa-miR-380-5p AK2 UCK1 RPS6KB2 hsa-miR-510 hsa-miR-383 hsa-miR-432* CAMK1 hsa-miR-193b RAF1 COL4A3BP AK7 CARKL ILKAP JAK2 NEK1 NME1 DGKG DUSP7 hsa-miR-409-5p AK5 hsa-miR-186 PANK1 SCYL1 TLR1 hsa-miR-153 hsa-miR-153 hsa-miR-101 hsa-miR-379 hsa-miR-185 hsa-miR-512-5p hsa-miR-517c hsa-miR-450 hsa-miR-431 DMPK hsa-miR-95 hsa-miR-208 PTPRR hsa-miR-192 hsa-miR-124a DKFZp434C1418 hsa-miR-7 hsa-miR-7 hsa-miR-378 hsa-miR-299-3p hsa-miR-363* CARD10 PAK2 LATS1 hsa-miR-30e-3p MAP3K14 hsa-miR-138 hsa-miR-138 DGUOK hsa-miR-511 RPS6KB1 hsa-miR-147 hsa-miR-199a* hsa-miR-199a* hsa-miR-188 hsa-miR-494 hsa-miR-504 TYK2 hsa-miR-452 PLK1
B
Fig. S4. Using FUSION mapping to identify miRNAs and kinases that regulate autophagy. (A) Unsupervised hierarchical clustering of reporter gene expression for miRNA mimics and
siRNAs. Regions containing siRNA targeting mTOR (upper) and ULK1 (lower) are enlarged.
(B) Density distribution of gene expression signatures of siRNA-miRNA pairs. Pairs with
identical seed regions (red, n = 311 pairs) show a larger average correlation than those with
different seed regions (black, n = 363,067 pairs). p = 5.1 × 10-9
, Wilcoxon rank-sum test. See
table S3 for a list of miRNA-siRNA pairs. (C) Flow cytometric measurement of GFP
fluorescence intensity in parental U2OS cells (blue curve) and in U2OS cells stably expressing
GFP-LC3 (red curve). Data are representative of five experiments. (D) GFP-LC3 fluorescence
intensity in U2OS cells transfected with the indicated siRNA pool. Data are representative of
five experiments. (E) The effect of the inhibition of autophagosome acidification with
Bafilomycin A1 (Baf A1) on GFP-LC3 fluorescence intensity in U2OS cells. Left: Flow
cytometric measurement of cells treated with Baf A1 (50 nM for 16 hours). Right: Median
fluorescence intensity (normalized to DMSO-treated and log2 transformed) as a function of time.
Data are representative of five experiments. (F) The effect of nutrient deprivation media (EBSS)
on GFP-LC3 fluorescence intensity in U2OS cells. Left: Flow cytometric measurement of cells
incubated for 2 hours in EBSS. Right: Median fluorescence intensity (normalized to untreated
control and log2 transformed) of cells exposed to EBSS in the presence or absence of Baf A1 (50
nM for two hours). Data represent five experiments.
Fig. S5. Representative flow cytometry data and characterization of the role of BMP2K in
autophagy and erythrocyte development. (A) GFP-LC3 fluorescence intensity in U2OS cells
transfected with the indicated miRNAs or siRNAs. Data are representative of three experiments.
(B) GFP-LC3 fluorescence intensity in U2OS cells transfected with individual siRNAs targeting
BMP2K. (C) Representative scatter plots from flow cytometry of mouse fetal liver cells before
(left) or after (right) purification of Ter119 negative erythroid progenitors, stained for Ter119
and CD71. (D) Representative scatter plots from flow cytometry of erythroid progenitor cells
infected with various shRNAs and stained for Ter119 and CD71. Regions indicated correspond
to lineages detailed in Fig. 3B.
Fig. S6. SRMS as an inhibitor of autophagy. (A) The effect of SRMS knockdown on GFP-
LC3 fluorescence. Representative images of GFP-LC3 in U2OS cellstransfected with siRNAs.
Data are representative of five experiments. (B) Quantification of GFP-LC3 fluorescence from
fluorescence microscopy images. Data are normalized to the intensity of GFP-LC3 in U2OS cells
transfected with a negative siRNA control (LONRF1). Mean + SEM. N = 3 experiments ***p <
0.001, unpaired t-test. NS, not significantly different from control. (C) The effect of SRMS
knockdown in the presence or absence of Bafilomycin A1. Left: Western blot of U2OS GFP-
LC3 cells transfected with siRNAs and treated with Bafilomycin A1 (BAF). Data are
representative of three experiments. Right: Quantification of flow cytometry of GFP-LC3 in
U2OS cells. Mean + SEM, N = 3 experiments. (D) GFP-LC3 punctae in U2OS cells transfected
with the indicated siRNAs. n = 51-149 cells per condition. Boxes represents median and
quartiles, whiskers extend to 1.5 × the interquartile distance, and outliers are indicated by circles.
*p < 0.05. ***p < 0.001, unpaired t-test. Data are representative of three experiments. (E) SRMS
knockdown does not affect mTOR activity. Western blot for the indicated proteins or
phosphoproteins in U2OS cells transfected with the indicated siRNAs. XPB is shown as a
loading control. LONRF1 is the negative control siRNA (Control). Data are representative of
three experiments.
Fig. S7. Using FUSION mapping to identify natural product inhibitors of autophagy and
migration. (A) Rank order plot of the similarity of reporter gene expression among crude natural
product fractions and siRNA knockdown of ULK1. Three red points highlight three different
crude fractions from a single organism, SN-B-019. (B) The SN-B-019 bacterium was recultured
and refractionated as described in Materials and Methods. Five pure compounds were isolated
and tested for their ability to inhibit autophagy. Left: Representative images of GFP-LC3 in
U2OS cells treated with the indicated purified compounds for two hours. Right: Quantification of
GFP-LC3 punctae in U2OS cells treated with the indicated purified compounds for two hours.
Mean; n = 20-74 cells per condition. Data are representative of two experiments. (C) Rank order
plot of the similarity of reporter gene expression among natural products and siRNA knockdown
of DDR2. (D) Representative images of the indicated cells treated with SN-A-048-7 (6.7 µg/ml)
or DMSO for five days. Data are representative of five experiments. (E) Dose response curve for
the effect of discoipyrrole A on inhibition of BR5 fibroblast migration from a collagen plug.
Mean; n = 3 fields. Data are representative of two experiments.
Fig. S8. Using FUSION to identify inhibitors of AKT signaling. (A) Left: Graph of the
similarity of reporter gene signatures produced by each natural product fraction as compared to
those for PDPK1 siRNA (x-axis) and TBK1 siRNA (y-axis) using the Mahalanobis distance sum
method. Right: Analysis of twelve natural product fractions for inhibition of AKT in HCC44
cells by Western blot. Data are representative of four experiments. (B) Analysis of the abundance
of the indicated proteins and phosphoproteins by Western blot of HCC44 cells treated with the
indicated natural product fractions for 2.5 hours. Data are representative of two experiments.
mapping
Sup. Figure 9 A
SNB-019-cmp1 (bafilomycin D)
1H NMR at 600 MHz
B
HRESIMS [M+H]
+ of SNB-019-cmp1 (bafilomycin D)
C
SNB-019-cmp2 (bafilomycin A1)
1H NMR at 600 MHz
D
HRESIMS [M+H]
+ of SNB-019-cmp2 (bafilomycin A1)
E SNB-019-cmp3 (bafilomycin B1)
1H NMR at 600 MHz
F
HRESIMS [M+Na]
+ of SNB-019-cmp3 (bafilomycin B1)
G
SNB-019-cmp4 (bafilomycin B2) 1H NMR at 600 MHz
H
HRESIMS [M+Na]
+ of SNB-019-cmp4 (bafilomycin B2)
I
Discoipyrrole A
1H NMR at 600 MHz in CD3OD
J
Discoipyrrole A
13C NMR at 600 MHz in CD3OD
K
HRESIMS [M-H]
– of discoipyrrole A
L
NMR data table for discoipyrrole A at 600 MHz in CD3OD
no. H, mult. (J in Hz) C
1 92.7
2 196.6
3 116.5
4 172.3
5 121.1
6, 6' 7.07, d (8.0) 131.7
7, 7' 6.85, d (8.0) 117.0
8 161.3
9 121.8
10, 10' 6.96, d (8.7) 131.6
11, 11' 6.63, d ( 8.7) 116.1
12 157.8
2.21, dd (14.1, 6.1) 43.1
1.99, dd (14.1, 6.5)
14 1.75, m 25.3
15 0.95, d (6.7) 24.4
16 0.84, d (6.7) 23.6
17 163.5
18 119.3
19 138.7
20 6.47, d (7.8) 123.6
21 7.43, ddd (7.8, 7.8, 1.4) 136.2
22 7.28, ddd (7.8, 7.8, 1.4) 126.4
23 8.03, dd (7.8, 1.4) 131.8
M
N SNB-003-cmp1
1H NMR at 600 MHz in DMSO-d6
O
N
O OO
O
O
O
Br
O
Br
HSQC of SNB-003-cmp1 at 600 MHz in DMSO-d6
Fig. S9. Natural product structure determination. (A) 1H NMR and of SNB-019-cmp1 at 600
MHz in CDCl3, matching structure of bafilomycin D. (B) HRESIMS of SNB-019-cmp1. (C) 1
H
NMR of SNB-019-cmp2 at 600 MHz in CDCl3 matching structure of bafilomycin A1. (D)
HRESIMS of SNB-019-cmp2. (E) 1H NMR of SNB-019-cmp3 at 600 MHz in CDCl3, matching
structure of bafilomycin B1. (F) HRESIMS of SNB-019-cmp3. (G) 1H NMR of SNB-019-cmp4
at 600 MHz in CDCl3, matching structure of bafilomycin B2. (H) HRESIMS of SNB-019-cmp4.
(I) 1H NMR of discoipyrrole A at 600 MHz in CD3OD (J)
13 C NMR of discoipyrrole A at 100
MHz in CD3OD. (K) HRESIMS of discoipyrrole A. (L) NMR data table for discoipyrrole A in
CD3OD. (M) Structure and X-ray crystal structure of the bis(p-bromobenzoate) derivate of
discoipyrrole A. Crystal structure deposited at CCDC, deposition # CCDC 910866. (N) 1H
NMR of SNB-003-cmp1 at 600 MHz in DMSO-d6. (O) 1H-
13C
HSQC of SNB-003-cmp1 at 600
MHz in DMSO-d6.
Fig. S10. Profiles of reporter gene expression changes in response to each of 1124 genetic
perturbations. Rank-ordered Log2 transformed normalized gene expression values for each
probe from one of three replicate measures.
Fig. S11. Analysis of correlation among reporter gene expression and between distance
metrics. (A) Normalized gene expression values for the 1,124 genetic perturbations from one of
three replicate measures is shown in a scatter plot of all possible pairs of the six reporter genes.
Relevant correlation coefficient (r) values are indicated in each plot. (B) Correlation between
Euclidean and Mahalanobis distance for all the possible pairs of 1,124 genetic perturbations.
Fig. S12. Estimation of FDR from value density distribution, rank sum as an example.
Empirical p values were estimated from the density distribution of the rank sum values, for each
pair of genetic perturbations from the permuted data set. The density distribution of the resulting
values is shown. The green curve indicates the fitted beta-uniform mixture model, and the
blue line indicates the null model. Please see Materials and Methods for more detail.
P
P
Table S1. Reporter gene expression in response to individual genetic and chemical
perturbations in HCT116 cells. (Tab 1) Legend for row and column annotations. (Tab 2)
genetic_all: rows 1-835 provide responses to each of 835 siRNA pools ( "siKinome" + replicated
control oligos); rows 836-1261 provide responses to each of 426 miRNA mimics (344 unique
miRNAs + redundant alleles); rows 1262-1274 provide responses to control siRNA oligos. (Tab
3) genetic_unique: rows 1-780 provide responses to each of 780 siRNA pools ("siKinome");
rows 781-1124 provide responses to each of 344 unique miRNA mimics (redundancy condensed
to mean of replicates). (Tab 4) chemical: responses to 1186 natural product fractions.
Table S2. Endogenous miRNA expression. Endogenous expression of miRNAs in HCT116
cells was measured in quadruplicate using the Human Illumina miRNA Array platform as
described in Materials and Methods.
Table S3. MiRNA-siRNA oligonucleotide seed identities. All instances in which an oligo
in an siRNA pool from the kinome siRNA library contains a seed sequence that matches a
miRNA seed sequence present in the miRNA mimic library are indicated. Also indicated is the
strength of similarity between the gene expression signatures produced by each pair of reagents
as defined by Pearson correlation and Euclidean distance.
Table S4. Kinase inhibition screen (KinaseProfiler). SNB-003-cmp1 was tested for inhibition
of kinase activity of 235 kinases at 3 nM and 0.3 nM concentrations, using the Millipore
KinaseProfiler service from Millipore as described in Materials and Methods. Data are presented
as percent activity.
Table S5. Maintenance and transfection conditions for cell lines used in this study.
