1
Supplemental Material
Supplemental Experimental Procedures
Fly genetics
Drosophila strains
Fly culture and crosses were performed according to standard procedures and were
raised at the indicated temperatures. Drosophila stocks used in this study are: E(spl)mγ-
GFP (S. Bray); Ase-GAL4 (T. Lee ); Erm-GAL4 (C.Y. Lee and G. Rubin); UAS-
aPKCCAAX (C.Q. Doe); Scabous-GAL4 (YN. Jan); 1407-GAL4 (L. Luo); lgl1 (F.
Matsuzaki); UAS-N, aph-1D35 (M. Fortini); UAS-TSC1, UAS-TSC2, UAS-PTEN (T. Xu);
UAS-dMyc (F. Demontis and B. Edgar); rheb2D1 (H. McNeill); meiP26fs1 (T. Cline);
spdoG104, ada1 (α-Adaptin6694), UAS-NΔECD, bratk06028, eIF4E-lacz (eIF-4E07238), eIF-
4ES058911, dMyc-lacZ [P{lacW}l(1)G0354G0354] (Mitchell et al. 2010), UAS-TSC1, UAS-
TSC-2 (T. Xu), Scabous-GAL4 (YN. Jan), UAS-4EBP(LL)s (N. Sonenberg), UAS-Flp;
Actin-FRT-stop-FRT-lacZ; UAS-GAL80ts, eIF4ES058911, UAS-Tor-DN, meiP26mfs1,
meiP261(Bloomington Drosophila stock center); UAS-eIF4E-RNAi (eIF4E-IR; #7800,
VDRC), UAS-eIF4E-RNAi-s (eIF4E-IR-s; HMS00969, TRiP), UAS-dmyc-RNAi
(#106066,VDRC), UAS-dmyc-RNAi-2 (#17487,VDRC), UAS-N-RNAi (#1112, #27229,
VDRC), UAS-Dicer2 (#60008, VDRC), UAS-polo-RNAi (#20177, VDRC), UAS-brat-
RNAi (HMS01121, TRiP). Note, for N knockdown, Dicer2 was coexpressed with N RNAi
to achieve efficient RNAi effects. All other common fly stocks were obtained from the
2
Bloomington Drosophila Stock Center, Szeged Drosophila Stock Center, the Vienna
Drosophila RNAi Center or the TRiP at Harvard Medical School.
Constructs
To generate HA-tagged dMyc and Flag-tagged heIF4E for coimmunoprecipitation
experiments, the open reading sequences of Drosophila dMyc and human eIF4E were
amplified by RT-PCR, and inserted into the pcDNA3-HA-N and pcDNA3-Flag-N vectors,
respectively. The accuracy of the PCR product sequence was confirmed by DNA
sequencing.
Chromatin immunoprecipitation
ChIP assays were based on previously described protocols (Krejci and Bray 2007;
Duan et al. 2011). In brief, yw 3rd instar larval brains were dissected in ice-chilled 1xPBS
and homogenized and fixed in 1% formaldehyde in buffer A [1.5 mM MgCl2, 10 mM KCl,
10 mM HEPES (pH7.9), 0.1% NP-40, protease inhibitor (Sigma)], for 10 min at room
temperature. After quenching, the tissue was washed three times with buffer A and
sonicated in buffer B (20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 2 mM EDTA, 1% Triton
X-100, 0.01% SDS, protease inhibitor) plus 1% SDS using Diagenode Bioruptor
Sonicator. Supernatant containing sheared chromatin of an average length of 0.5 kb
was diluted with buffer B, precleared with BSA-coated protein A or G beads and
incubated overnight at 4°C with anti-Su(H) (Krejci and Bray 2007) or anti-dMyc (Maines
3
et al. 2004; Teleman et al. 2008) antibodies. ChIP performed in parallel with normal
goat or rabbit IgG (Santa Cruz Biotechnologies) served as negative controls. After
extensive washing, elution and reverse crosslinking, precipitated DNA fragments were
purified using Qiagen spin columns and eluted in 30 µl elution buffer. Real time PCR
analysis was performed on a StepOnePlus real time PCR system (Applied Biosystems)
using SYBR Green PCR Master Mix (Applied Biosystems). Results were quantified
using the ΔCt methods, with respect to input samples. Primer sequences are available
upon request.
Coimmunoprecipitation
HEK293T nuclear extracts were prepared as described (Dignam et al. 1983) with some
modifications. Briefly, HEK293T cells were suspended in hypotonic buffer (10 mM
HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, protease inhibitor) and incubated on ice
for 15 min. After cell lysis and centrifugation, the supernatant was saved as the
cytoplasmic fraction, while the crude nuclei pellet resuspended in Nuclear Extraction
buffer [20 mM HEPES (pH 7.8), 0.4 M KCl, 1.5 mM MgCl2, 0.2 mM EDTA (pH 8.0), 25%
glycerol, and protease inhibitors], incubated on ice for 30 min and sonicated using
Diagenode Bioruptor Sonicator for 3 cycles. The nuclear extracts were clarified by
centrifugation, and proteins immobilized by binding to anti-Flag M2 affinity gel (A2220,
Sigma-Aldrich) for 4 hr or overnight at 4°C. Beads were washed and proteins recovered
directly in SDS-PAGE sample buffer. Rabbit anti-Flag (F7425, Sigma-Aldrich), rabbit
4
anti-c-myc (A-14, Santa Cruz Biotechnologies) or rabbit anti-HA (71-5500, Zymed) were
used for western blot analysis.
eIF4E inhibitor treatment
Ribavirin (Sigma-Aldrich) was added to standard fly food at 500 µM final concentration.
Embryos of various genotypes were collected on Ribavirin-containing food or standard
fly food (served as control) for 6 hr at 25°C and allowed to develop to 120 hr ALH before
larval brain dissection and immunostaining.
Analysis of ovaries
For rescue of mei-P2mfs1/1 mutant ovarian tumor phenotypes, mei-P26mfs1/Basc;
eIF4ES058911/TM2 or mei-P26mfs1/Basc; rheb2D1/TM2 females were crossed to mei-
P261/Y males. Newly eclosed F1 females of mei-P26mfs1/1; eIF4ES058911/+ or mei-
P26mfs1/1; rheb2D1/+ genotypes were fed with dry yeast for 3 days before ovary
dissection. Sibling females of the mei-P26mfs1/1; TM2/+ genotype served as internal
control. For the rescue of mei-P2fs1mutant ovarian tumor phenotypes, mei-P26fs1/Basc;
eIF4ES058911/TM2 or dMycG0354, mei-P26fs1/FM6B females were crossed to mei-P26fs1/Y
males. Newly eclosed F1 females of mei-P26fs1; eIF4ES058911/+ or dMycG0354, mei-
P26fs1/mei-P26fs1 genotypes were fed with dry yeast for 3 days before ovary dissection.
Sibling females of the mei-P26fs1; TM2/+ genotype served as internal control.
5
Inducible expression experiment
Embryos of 1407-GAL4, tub-GAL80ts>N-IR, Dicer2 or 1407-GAL4, tub-GAL80ts >N-IR,
Dicer2; dMyc genotypes were collected for 4-6 hr and allowed to develop at 22°C
(permissive temperature). Larvae at 42 hr ALH were shifted to 29°C (restrictive
temperature) and processed at indicated time points ATS.
Conditional rescue experiment
In order to effectively knockdown eIF4E after the brain tumor phenotype has been
developed in brat mutants, a stronger RNAi line, eIF4E-RNAi-s, was used in this
analysis (Fig. S10). Embryos from cross between1407-GAL4, brat/Bc-Gla; tub-GAL80ts
flies and brat/Bc-Gla; UAS-eIF4E-IR-s flies were collected for 4-6 hr at 22°C and
allowed to develop to embryo-hatching stage. Newly-hatched larvae were raised at
18°C for 112 hr before being shifted to 29°C. Dissection I, II and III (DI, DII and DIII)
were performed at 24 hr, 48 hr and 56 hr after the 29°C shift.
6
Supplemental Figure Legends
Figure S1. Evidence supporting selective regulation of Type II but not Type I NB by N
Signaling. (A, B) UAS-N RNAi (N-IR) was induced by the 1407ts system (1407-GAL4;
tub-GAL80ts). Larvae were shifted to 29°C at 42 hr ALH and larval brains were analyzed
at indicated time points after temperature shift (ATS). At 38 hr ATS, the cell size of type
II NBs expressing N-IR was greatly reduced. At 48-52 hr ATS, type II NBs were no
longer detectable in the type II lineages. The cell sizes of type I NBs (yellow arrowheads)
remained constant over time. Quantification of NB size is shown in (B). From this panel
on, NBs are marked with white brackets. (C) A diagram of type I NB lineage. Type I NB:
Dpn (red)+, cytoplasmic Pros (blue); GMC or neurons: Dpn-, nuclear Pros. A type I NB
undergoes asymmetric division to self-renew and give rise to a GMC, which divides one
more time to produce two terminally differentiated neurons. (D) Expression of N reporter
E(spl)mγ-GFP (green) in type I NB lineage. Dlg staining outlines the cell cortex. (E) Cell
size of type II but not type I NBs was reduced in aph-1 mutant NB clones at 72 hr after
clone induction (ACI). Scale bar, 10 µm (A, E).
Figure S2. Evidence supporting cell growth defects as a primary consequence of N
inhibition in NBs. (A) The intensity of Pros levels in WT or spdo mutant NBs were
measured at different time points ACI and then normalized with Pros levels in WT
mature IPs present outside of the induced mutant clones within the same images. (B)
Clonal analysis of type II NBs of spdo single or spdo, pros double mutant at 52 hr ACI.
Green: GFP; red: Dpn; blue: Pros. (C) Clonal analysis of type II NBs of aph-1 mutant at
7
various time points ACI. Green: GFP; red: Ase; blue: Pros. Mature IPs were marked
with closed arrowheads. Scale bar, 10 µm (B, C).
Figure S3. N signaling is overactivated in ada mutant NBs. (A) Expression of N reporter
E(spl)mγ-GFP (green) in WT or ada mutant larval brain showing its upregulation in ada
mutant NBs. (B) The NB overproliferation phenotype in ada mutants was completely
rescued by Notch knockdown, suggesting that Ada may normally inhibit ectopic NB
formation by downregulating N signaling. (C) Quantification of data from (B). Scale bar,
100 µm (B); 10 µm (A).
Figure S4. Ectopic NB formation induced by overactivation of N signaling observed
over multiple time points. (A) Ectopic NBs [Dpn+, Pros-, yellow arrowheads] formed in
ada mutant clones at various time points ACI. At 30 hr ACI, ada mutant clones
contained a single primary NB, which was in direct contact with immature IPs, followed
by mature IPs and neurons. At 48 hr ACI, ectopic NBs of smaller cell size than the
primary NB (4-6µm in diameter) were found further away from the primary NB than
immature or mature IPs. At 70 hr ACI, ada mutant clones contained a chain of ectopic
NBs of increasing cell sizes. The largest ectopic NB (≥10 µm, yellow bracket),
morphologically indistinguishable from a normal NB, was farthest away from the primary
NB. At 90 hr ACI, while immature IPs were adjacent to the primary NB, ~10 full-sized
ectopic NBs (≥10 µm, yellow bracket) formed several cell diameters away from the
8
primary NB. From this panel on, yellow arrowheads mark smaller-size ectopic NBs,
while yellow bracket mark full-sized ectopic NBs (≥10 µm). (B) Ectopic NBs formed in
clones induced by a constitutively active form of N (Nact) at various time points ACI.
While no ectopic NB was found in Nact clones at 30 hr ACI, Nact clones contained more
ectopic NBs than ada mutant clones at 48 hr ACI, presumably due to the fact that
constitutive activation of N is more potent than loss of Ada in inducing dedifferentiation
of IPs back into ectopic NBs. At 70 hr ACI, 6-10 full-sized and numerous intermediate-
sized ectopic NBs were found in the clones. (C) Quantification of total (≥6 µm, green) or
full-sized (≥10µm, purple) ectopic NBs in ada mutant of Nact clones over multiple time
points. Scale bars, 10 µm.
Figure S5. Effects of TOR pathway inhibition on brain tumor formation. (A) The effects
of knocking down eIF4E or overexpressing negative regulators of TOR pathway,
TSC1/2, 4EBP(LL)s or a dominant-negative form of TOR kinase, TOR.TED, on NB
overproliferation in various brain tumor backgrounds. (B) Quantification of data from (A).
Scale bar, 100 µm.
Figure S6. eIF4E knockdown specifically suppressed ectopic NB formation in the Type
II lineage. (A) eIF4E knockdown completely suppressed the brain tumor phenotypes
caused by aPKCCAAX overactivation or brat mutation. Posterior surface views of whole
brains are shown (green, NBs marked by Dpn; red, neurons marked by Pros). White
9
dotted line indicates the boundary between the optic lobe (lateral) and the central brain
(medial) areas. (B, C) eIF4E RNAi within a clone (driven by Elav-GAL4) efficiently
knocked down eIF4E protein expression, resulting in undetectable eIF4E expression
within the clone (NBs are marked with stars), but it had no discernable effects on either
the maintenance or the composition of a type II NB lineage. (D) Specific knockdown of
Polo kinase or Pros in type I lineages, driven by Ase-GAL4 (Bowman et al. 2008), led to
ectopic type I NB formation. Such phenotypes were not affected by eIF4E RNAi.
Anterior views of single brain lobes are shown (green, Dpn). (E) Quantification of data
from D (n=15-20). (F) Notch overactivation in SOP (driven by Sca-GAL4) led to cell fate
transformation and resulted in a multi-socket phenotype. Such phenotype was not
affected by eIF4E RNAi. Blue arrowhead: socket; yellow arrowhead: bristle. (G) A model
depicting higher dependence on eIF4E by ectopic type II NBs than WT type II NBs, or
ectopic type I NBs in cnn or polo mutant. Scale bars, 10 µm (B,C); 100 µm (D).
Figure S7. Supporting evidence for a regulatory loop involving eIF4E and dMyc. (A)
Ectopic NB formation due to ada mutation or N overactivation was suppressed by eIF4E
RNAi or dMyc RNAi. Importantly, the tumor suppression effects conferred by eIF4E
knockdown were partially abolished when dMyc but not GFP was overexpressed. Green,
Dpn; red, Pros. (B) Quantification of data shown in (A). * p<0.0001 (n=10-15). Scale
bars, 100 µm (A).
10
Figure S8. Overproliferation of NBs in various brain tumor backgrounds was effectively
suppressed by eIF4E RNAi. (A) Proliferative ability of NBs in various backgrounds was
assayed by PH3 staining. Green: Mira; red: PH3. (B) eIF4E or dMyc knockdown
showed no discernible effects on the apical-basal polarity of WT NBs. Green: Mira; red:
PH3. (C) Quantification of dividing NBs in various backgrounds as examined in (A). *
p<0.003 (n=8-10). Scale bar, 100 µm.
Figure S9. Conditional suppression of NB overproliferation in brat mutants by eIF4E
RNAi. (A) Expression of eIF4E-IR-s, a strong RNAi line for eIF4E, was induced when
GAL80ts was inhibited after a temperature shift from the permissive temperature (18°C)
to restrictive temperature (29°C). DI, DII and DIII indicate brain dissections at three
consecutive time points following temperature shift. Quantification of data is shown in
(B). * p<0.001 (n=5-10). Scale bars, 100 µm (A).
Figure S10. eIF4E protein levels in type II NBs of various genetic backgrounds. (A) A
type II NB lineage in each background was delineated by yellow dotted line and its
primary NB marked with a star. Relative eIF4E fluorescence indicated the ratio of eIF4E
intensities within NBs and an area encircled by blue dashed line, where 1407-GAL4
expression was lacking. **p<0.0001; *p<0.02 (n=15-20). Scale bars, 20 µm (A).
11
Figure S11. Evidence supporting specific suppression of ectopic NBs by dmyc RNAi. (A)
dMyc knockdown using dmyc-IR-2 RNAi line effectively suppressed ectopic NBs
induced by N. Quantification of data is shown in (B). Scale bars, 100 µm (A).
Figure S12. eIF4E promoter activity is specifically reduced by eIF4E RNAi. (A)
Expression of dMyc protein in various backgrounds. (B) Expression of the dMyc-lacZ
reporter in various backgrounds. (C) Expression of the eIF4E-lacZ reporter in WT or
eIF4E knockdown backgrounds. Scale bars, 10 µm (A-C).
Figure S13. Supporting evidence that dMyc-mediated cell growth is important for
maintaining NB stem cell identity. (A) A control experiment for Fig. 5C, showing that co-
overexpression of a UAS-CD8-GFP transgene with N-IR failed to prevent type II NB
elimination induced by N knockdown, suggesting N-IR effect is not sensitive to added
UAS transgene expression. (B,C) UAS-N RNAi (N-IR) alone (middle) or N-IR plus dMyc
(right) were induced with the 1407ts system. Larvae were shifted to 29°C at 42 hr ALH
and larval brains were analyzed at 44 hr after temperature shift. While NBs were no
longer detectable in the type II lineages of N-IR-expressing animals, coexpression of
dMyc significantly prevented such NB loss. Quantification of type II NB number in WT,
1407ts>N-IR or 1407ts>N-IR; dMyc genotypes is shown in (C). **p<0.0005 (n=15-20).
Scale bars, 10 µm (B); 50 µm (A).
12
Figure S14. Ribavirin treatment showed no effect on WT NB maintenance. (A) WT
larval brain treated with or without 500 µM Ribavirin. Quantification of type II NB number
is shown in (B). Scale bars, 100 µm (A).
Figure S15. A model depicting the regulation of normal stem cells and CSCs by the
eIF4E-dMyc regulatory loop in the larval brain (A) or the ovary (B). (A) Differential N
signaling and Brat determines NSC vs. IP cell fate. In NSCs, N signaling, positively
regulated by Spdo and Aph-1 (not shown), promotes the eIF4E/dMyc-mediated stem
cell growth and self-renewal through a transcriptional cascade. Within IP cells, N
signaling is inhibited by the α-Adaptin (Ada) and Numb complex. As a consequence, IPs
reduce their cell growth, cell size, and commit to differentiation. Brat also restricts IP cell
growth by suppressing dMyc expression. Loss of Brat or overactivation of N relieves the
restriction on eIF4E-dMyc in IPs, which grow faster than normal NSCs and
dedifferentiate into ectopic NSCs. Conversely, N inhibition or knockdown of both eIF4E
and dMyc slows down cell growth of NSCs, which gradually reduce in size and
eventually lose their stem cell fate. Dark or light color of each component indicates its
high or weak activity, respectively. For further description, see Discussion. (B)
Differential Mei-P26 levels determine GSC vs. cystocyte cell fates. In GSCs, the eIF4E-
dMyc axis promotes stem cell growth and self-renewal. Within cystocytes, Mei-P26
restricts cell growth, by directly or indirectly suppressing dMyc expression. Loss of Mei-
P26 relieves the restriction on eIF4E-dMyc in cystocytes, which then grow faster and
dedifferentiate into ectopic CSCs. Conversely, Mei-P26 overexpression would slow
13
down the cell growth of GSCs, which would eventually lose their stem cell fate. Dark or
light color of each component indicates high or low activity, respectively.
References
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2008. The tumor suppressors Brat and Numb regulate transit-amplifying
neuroblast lineages in Drosophila. Dev Cell 14(4): 535-546.
Dignam, J.D., Lebovitz, R.M., and Roeder, R.G. 1983. Accurate transcription initiation
by RNA polymerase II in a soluble extract from isolated mammalian nuclei.
Nucleic Acids Res 11(5): 1475-1489.
Duan, H., Dai, Q., Kavaler, J., Bejarano, F., Medranda, G., Negre, N., and Lai, E.C.
2011. Insensitive is a corepressor for Suppressor of Hairless and regulates Notch
signalling during neural development. EMBO J 30(15): 3120-3133.
Krejci, A. and Bray, S. 2007. Notch activation stimulates transient and selective binding
of Su(H)/CSL to target enhancers. Genes Dev 21(11): 1322-1327.
Maines, J.Z., Stevens, L.M., Tong, X., and Stein, D. 2004. Drosophila dMyc is required
for ovary cell growth and endoreplication. Development 131(4): 775-786.
Mitchell, N.C., Johanson, T.M., Cranna, N.J., Er, A.L., Richardson, H.E., Hannan, R.D.,
and Quinn, L.M. 2010. Hfp inhibits Drosophila myc transcription and cell growth
in a TFIIH/Hay-dependent manner. Development 137(17): 2875-2884.
14
Teleman, A.A., Hietakangas, V., Sayadian, A.C., and Cohen, S.M. 2008. Nutritional
control of protein biosynthetic capacity by insulin via Myc in Drosophila. Cell
Metab 7(1): 21-32.
Song_Fig. S1
F-actin Dpn ProsA
38 hr 52 hr
B
ete
r (
M)
C D GFP Dpn ProsE
WT N-IRN-IR
NB
dia
me
Hours after 29C shift
0 38 52
GMC
Neurons
Type I NB
C D
Dpn E(spl)m Dlg
GFP p
72 hr
aph-1Type II Type I
E72 hr
aph-1p p
DpnGFP: Prosce spdo NBs
WT NBsA B
Song_Fig. S2
spdo
52 hr 52 hr
spdo; pros
28
48
72 28 45 48 IPs
Hours after clone induction
Nor
mal
ized
P
ros
fluor
esce
n c spdo NBs
WT mature IPs
GFP Ase ProsC
48 hr 60 hr 70 hr 74 hr Type I 74 hr
aph-1 aph-1 aph-1aph-1 aph-1
A
Song_Fig. S3
Dpn ProsE(spl)m
WT
ada
B Mira
ada ada; N RNAiWT
ada
ada; N RNAi
WT
NB
nu
mb
er
pe
r b
rain
lob
e
C
*
ada ada; N RNAiWT
30 hr 48 hr 48 hr GF
P
A
Song_Fig. S4
ada ada
90 hr P
Dpn
Pros
ada
70 hr
0m -1.8m
30 h 48 h
adaadaada
B
0m -0.8m
NN
30 hr 48 hr
70 h0 3 2 m
N
70 hr GF
PD
pnP
ros
0m -3.2m
# Ectopic NBs (≥6m in diameter)
# Ectopic NBs (10m in diameter)
ber
per
clon
e
C
NB
num
b
MiraA
Song_Fig. S5
WT Tor-DN4EBP(LL)sTSC1/2eIF4E-IR
ada ada, 4EBP(LL)s ada, Tor-DNada, TSC1/2ada; eIF4E-IR
brat brat; 4EBP(LL)s brat-IR; Tor-DNbrat-IR; TSC1/2 brat; eIF4E-IR
N; Tor-DNN N; 4EBP(LL)sN; TSC1/2N; eIF4E-IR
B
+eIF4E-IR
+TSC1/2
+4EBP(LL)s
+Tor-DN (TED)
Control
NB
nu
mb
er
pe
r b
rain
lob
e
B
WT ada brat N
A Dpn Pros
Song_Fig. S6
aPKCCAAX aPKCCAAX, eIF4E-IR brat brat; eIF4E-IR
GFP eIF4E Pros
a
* *
**
** *
**
*
B C GFP Dpn Pros
**
eIF4E-IReIF4E-IR eIF4E-IRWT
DpnD
control+eIF4E-IR
polo-IRWT
E
#N
B/ b
rain
lob
e
NS
NS
NSSOP
F
GWT cnn or polo N or brat
polo-IR; eIF4E-IReIF4E-IReIF4E-IR
#
WT N N;4E-IR
Higher dependence on eIF4E
Song_Fig. S7
Dpn ProsA
ada; eIF4E-IRadaada; eIF4E-IR; dMyc
ada; eIF4E-IR; GFP ada; dmyc-IR ada; dMyc
B
mb
er
lob
e *
N N; dmyc-IRN; eIF4E-IRN; eIF4E-IRdMyc N; dMyc
+eIF4E-IR
+ IF4E IR dM
Control
N; eIF4E-IRGFP
NS
NB
nu
mp
er
bra
in
ada N
*+eIF4E-IR, dMyc
+dmyc-IR
+dMyc
N; GFP
+eIF4E-IR, GFP
+GFP
NS NS
PH3MiraA
Song_Fig. S8
bratWT aPKCCAAX
brat; eIF4E-IReIF4E-IR aPKCCAAX; eIF4E-IR
B
dmyc-IR dmyc-IR; eIF4E-IR
CWT
dmyc-IRWT
eIF4E-IR
s * *
1407>eIF4E-IR
1407>dmyc-IR
1407>eIF4E-IR; dmyc-IR
1407>aPKCCAAX
WT
1407>aPKCCAAX; eIF4E-IR
brat
brat; 1407>eIF4E-IRNu
mb
er o
f M
ph
ase
NB
pe
r b
rain
lob
e
*NS
NS
MiraA
D I D II D III D III
Song_Fig. S9
brat brat brat
D I D II D III
WT
D III
brat; 1407ts>4E-IR-s brat; 1407ts>4E-IR-s brat; 1407ts>4E-IR-s 1407ts>4E-IR-s
B
NB
num
ber
per
brai
n lo
be
*
*
**
NS
D II
D III
1407ts>4E-IR-s
WT
D I
Song_Fig. S10
ProseIF4EA
*
*
*
WT eIF4E-IR eIF4E-IR -s
**
*Rel
ativ
e F
4Eflu
ores
cenc
e
WT
eIF4E-IR
eIF4E-IR-s
dmyc-IR; eIF4E-IR
B*
WT eIF4E IR eIF4E IR s
eIF
dmyc-IR; eIF4E-IR
A ProsDpn
Song_Fig. S11
dmyc-IR -2 N; dmyc-IR -2N
control
+ dmyc-IR-2
Typ
e II
NB
num
ber
per
brai
n lo
be
WT
NS
B
N
NB
num
ber
per
brai
n lo
be
N
dMycF-actinA
eIF4E
WT
Myc ce
*
Song_Fig. S12
dMyc-lacZ F-actinB
WT eIF4E-IR dMyceIF4E
e
eIF4E-IR
dMyc
Rel
ativ
e dM
fluor
esce
n
NS
NS
WT dMyceIF4E eIF4E-IR dmyc-IR
tive
-lacZ
scen
ce eIF4E
eIF4E-IR
WTNS
NS
NS
eIF4E-lacZF-actin Pros
eIF
4E-la
cZsc
ence
*eIF4E-IR
WT
C
Rel
adM
yc-
fluor
es dMyc
dmyc-IR
eIF4E-IRWT
Rel
ativ
e e
fluor
es
F-actin Dpn ProsA
Song_Fig. S13
N-IR N-IRN-IR; GFP
N-IR; GFP
NB
line
ages
rain
lobe
**
F-actin Dpn Pros
44 hr 44 hr
B C
#typ
e II
NP
er b
WT
N-I
R
N-I
R; d
Myc
WT N-IR N-IR; dMyc
Dpn ProsA
Song_Fig. S14
B
500 µM
WT
0 µM
WT 0 M
500 M
Typ
e II
NB
num
ber
per
brai
n lo
be
WT
NS
Song_Fig. S15
CC
A B