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

Bowman, S.K., Rolland, V., Betschinger, J., Kinsey, K.A., Emery, G., and Knoblich, J.A.

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