Early 3rd (72h)
1.0
0.8
0.6
0.2
E74
0.0
mtl rRNA
)
La
te
3r
d
d
3r
id
M
(1
19
6h
(9
(7
d
3r
rly
Ea
h)
BR-C
)
0.4
2h
Relative pri-miR-8 level
1.2
Late 3rd (119h)
RT-PCR
Mid 3rd (96h)
qRT-PCR
Supplementary Figure 1. The level of pri-miR-8 gradually decreases while those of BR-C
and E74 increase during 3rd instar larval development. RNA was prepared from wild type
whole larva (w1118) at indicated time point (AEL) and levels of pri-mir-8, BR-C, and E74 were
analyzed by qRT-PCR and semi-quantitative RT-PCR. Mitochondrial large ribosmal RNA1 (mtl
rRNA1) was used for normalization. This experiment was independently repeated with similar
result.
Northern Blot
20E
72h
EtOH
20E
48h
EtOH
20E
24h
EtOH
20E
EtOH
16h
20E
EtOH
6h
20E
EtOH
3h
miR-8
miR-8/
2S rRNA 100 99 100 91 100 63 100 68 100 49 100 51
2S rRNA
1
2
3
4
5
6
7
8
9
10 11 12
Supplementary Figure 2. Treatment of 20-hydroxyecdysone (20E) in S2 cells progressively decreases the miR-8 levels analyzed by northen blot. Ethanol (EtOH) is a vehicle for
20E. 2S rRNA is a loading control used for miR-8 quantification. Times post 20E/EtOH treatment are denoted above the blot.
position (strand)
sequence
factor name
-2778 (+)
-2520 (+)
-2516 (+)
-2356 (+)
-2204 (-)
-2167 (-)
-1963 (-)
-1544 (+)
-1503 (+)
-1093 (+)
-718 (+)
-632 (+)
-456 (+)
-402 (+)
113 (+)
128 (+)
418 (+)
437 (-)
516 (-)
539 (+)
557 (+)
aaaaacAGGAAaagggt
ttaaacGGGAAagacaa
acgggaaagACAAGaata
taattatatACAAAcaca
ttattTTTACtat
tttttTTTTTtaa
tttatTTTAAaaa
aatAGAAAtaaaa
taaATAAAccaaa
aaattatagGCAATtaag
tttttattaCCAAAttcg
taaccatgaACAAGtctt
ccaaaaAGGAAgccgtg
agttaaaaaTCAAAtcac
ataGTAAAataaa
ataGAAAAcagaa
tcaATAAActaat
tggcTTTGTttttgtttta
ttaaaTTTACaaa
aagcgacaaACAAAattc
aaaAGAAAgaaaa
E74A
E74A
BR-C Z1
BR-C Z1
BR-C Z4
BR-C Z4
BR-C Z4
BR-C Z4
BR-C Z4
BR-C Z1
BR-C Z1
BR-C Z1
E74A
BR-C Z1
BR-C Z4
BR-C Z4
BR-C Z4
BR-C Z1
BR-C Z4
BR-C Z1
BR-C Z4
Supplementary Figure 3. Predicted binding sites of E74 and BR-C in promoter region of
miR-8. Numerous binding sites of E74 and BR-C were predicted in promoter region of miR-8
analyzed by P-Match and Match programs. Candidates were obtained by minimizing both
false-negative and false-positive.
Cloned gDNA
PR1-miR-8
PR2-miR-8
-2884
-1252
-2884
TF binding site:
CG34460
+760
BR-C
E74
mir-8
Supplementary Figure 4. Schematic representation of promoter region of miR-8. Two
genomic DNA fragments (PR1-miR-8 and PR2-miR-8) were cloned into each of the pGL3
luciferase reporters. Putative binding sites of E74 and BR-C are shown. +1 indicates the
pri-miR-8 transcription start site as previously described [Qian J, Zhang Z, Liang J, Ge Q,
Duan X, et al. (2011) The full-length transcripts and promoter analysis of intergenic microRNAs
in Drosophila melanogaster. Genomics 97: 294-303].
Luciferase Assay
EtOH
20E
Relative expression (FL/RL)
1.2
p=0.01
p=0.03
1.0
0.8
0.6
0.4
0.2
0.0
pGL3
null
pGL3 pGL3 bm1&2
PR1-miR-8 PR1-miR-8
Supplementary Figure 5. Mutagenesis of predicted E74 binding sites in pGL3 PR1-miR-8
reporter. Mutations of first two predicted binding sites of E74 (bm1&2) in pGL3 PR1-miR-8
reporter partially derepress the suppressive effect of 20E on reporter activity (n=4). p values
are denoted for the significance. Error bars denote SEM.
Timing of Puparium Formation
112.2 +/- 6.0
armG4/+
115.3 +/- 8.2
arm>EcR i
124.9 +/- 7.2
cgG4/+
129.6 +/- 8.0
cg>EcRDN
0
20
40
60
AEL (hr)
80
100
120
140
Supplementary Figure 6. Pupariation timing of the flies in which EcR activity is
inhibited. Ubiquitous knockdown of EcR (arm>EcR i) or expression of dominant negative EcR in
fat body (cg>EcRDN) marginally delays pupariation time. Mean times of the pupariation in each
of the flies are shown. More than 222 pupae in each of the genotype are counted. Error bars
mean SD.
qRT-PCR
Relative dMyc level
1.5
1.2
0.9
0.6
0.3
N
D
>E
cR
cg
cg
G
4/
+
0.0
Immunlostaining
CD2
dMyc
Merged
EcRDN
OE
cells
Supplementary Figure 7. EcRDN marginally affects the dMyc expression in fat body of
mid 3rd instar larva. (Top) expression of EcRDN in larval fat body has little effect on mRNA
level of dMyc shown by qRT-PCR. Four biological replicates in each genotype were examined.
Error bars denote SEM. (Bottom) Fat cells clonally expressing EcRDN (marked by absence of
CD2) express dMyc protein with similar levels as neighboring wild type cells.
Pupal Volume
140
p=0.0001
p=0.01
120
Relative size
100
80
60
40
20
h
us
R
us
h>
Ec
us
R
h>
i&
Ec
us
us
h>
4/
hG
us
i
h
+
0
Supplementary Figure 8. Overexpression of USH impedes the increase of pupal size
induced by EcR knockdown. Knockdown of EcR in the tissues where endogenous USH is
expressed increases the final pupal size, which effect is impeded by overexpression of USH.
More than 20 pupae in each genotype were measured. p values are denoted for the significance.
Error bars denote SEM.
Supplementary Materials and Methods
Quantitative RT-PCR
RNA was extracted with the Trizol reagent (Invitrogen). Analysis was performed on
an ABI7300 instrument with ABI SYBR PCR master mix (Applied Biosystems). primiR-8 and mRNA levels were calculated as a relative fold-change over rp49 mRNA
except as otherwise indicated. The comparative cycle threshold (Ct) method was applied
to estimate mRNA levels. The following PCR primers were used: pri-mir-8, 5′-GCT
TCC TCT GGC AAA GGA GAT T-3′ and 5′-GGA TCT AAT GCT GCC CGG TAA3′; ush, 5′-TCG AAT TCC GAT GTA TCC AAC G-3′ and 5′-GGA GTT GTT GTT
CTC GTG CAC C-3′; Inr, 5′-GGA TGC GTG ATC GAT AAG AA-3′ and 5′-CTG
ATA ATA GCC TTT CGG ACA G-3′; step, 5′-GAC AAA CCC ACC GTT GAT C-3′
and 5′-GCT CTG TTC GAA TTG ACT CG-3′; myc, 5′-CAG TTC CAG TTC GCA
GTC AA-3′ and 5′-AGA TAA ACG CTG CTG GAG GA-3′; rp49, 5′-AGG GTA TCG
ACA ACA GAG TG-3′ and 5′-CAC CAG GAA CTT CTT GAA TC-3′; and
mitochondrial large ribosomal RNA1 (mtl rRNA1), 5′-AAA AAG ATT GCG ACC
TCG AT-3′ and 5′-AAA CCA ACC TGG CTT ACA CC-3′.
Northern blot
Larval fat bodies were dissected in PBS and immediately transferred to Trizol
(Invitrogen). RNA was loaded and resolved on a 12.5% urea-polyacrylamide gel and
transferred electronically to Zeta-probe GT membrane (Biorad). The oligonucleotide
complementary to miR-8 or 2S rRNA was end-labeled with [γ-32ATP] and used as a
probe for northern blotting. The following oligonucleotides were used as the probe.
miR-8, 5′-GAC ATC TTT ACC TGA CAG TAT TA-3′; and 2S rRNA, 5′-AAC CCT
1
CAA CCA TAT GTA GTC-3′. The membrane was exposed to a phosphor imaging
plate (Fujifilm) and read with the BAS-2500 system (Fujifilm).
Immunostaining and clonal analysis
Mid 3rd instar larvae (~98 h AEL) were dissected and fixed in 4% formaldehyde and
blocked as previously described (Hyun et al. 2009). Antibodies against CD2 (1:200,
Serotec) were used. The antibody against USH (1:500) was described elsewhere (Hyun
et al. 2009). Anti-dMyc antibody was purchased from Santa cruz. Anti-mouse and antirabbit IgG Alexa 488/594 secondary antibodies (1:200, Molecular Probes) were used.
The images were obtained with a Zeiss LSM510 confocal microscope. For inducing the
FLP-out GAL4 overexpression clone in fat bodies, 1st instar larvae (1 day AEL) were
heat shocked at 39°C for 10 min.
Western blot
To detect phosphorylated Drosophila Akt, the fat bodies of mid 3rd instar larvae (~98 h
AEL) were dissected and homogenized in 120 μL lysis solution (1% Triton X-100, 50
mM Tris pH 7.4, 500 mM NaCl, 7.5 mM MgCl2, 0.2 mM EDTA, 1 mM NaVO4, 50
mM NaF, 1 mM DTT, and 25% glycerol) as previously described (Hyun et al. 2009).
ImmobilonTMPVDF Membrane (Millipore) was used for western blotting. Before
blocking in 4% BSA, the membrane was dried for 30 min at 37°C. The membrane was
then incubated overnight at 4°C with anti-phospho-Akt (Ser505, Cell Signalling) or
anti-Actin (Santa Cruz) antibodies.
Plasmid construction
2
The reporter plasmid containing the predicted promoter region of miR-8 was generated
by inserting the PCR-amplified promoter sequences upstream of the luciferase gene in
the pGL3 basic vector (Promega). The following primers were used to amplify the
promoter fragments of miR-8: PR1-miR-8, 5′-GCC GGC ATA TTT CTG GTT TTC
GTG T-3′ and 5′-GTG CTG GGT GCT GAG TGC TG-3′; and PR2-miR-8, 5′-GCC
GGC ATA TTT CTG GTT TTC GTG T-3′ and 5′-GGA AGC TCA AAG TTC TCA
AGG CGA-3′. The first two predicted binding sites of E74 in pGL3 PR1-miR-8 reporter
were mutated by using the QuickChange Site-Directed Mutagenesis kit (Stratagene).
The primer sets were as follows: bm1, 5′-CAA ATC GGC CCA AAA AAC ATT CCA
AGG GTT TCT TCG ATC GG-3′ and 5′-CCG ATC GAA GAA ACC CTT GGA ATG
TTT TTT GGG CCG ATT TG-3′; and bm2, 5′-GCA TGA TGT ACA TTA AAC GTT
CCA GAC AAG AAT AAA CCT CC-3′ and 5′-GGA GGT TTA TTC TTG TCT GGA
ACG TTT AAT GTA CAT CAT GC-3′.
Knockdown experiment in S2 cells
The MEGAscript T7 kit (Ambion) was used to generate dsRNAs. S2 cells were treated
twice with 12 μg dsRNA on the first and third days. The cells were treated with 10 μM
20E or with an equal volume of EtOH for 6 h on the fourth day. To produce templates
for T7 transcription, the following primers were used for PCR: GFP, 5′-TAA TAC
GAC TCA CTA TAG GGA GAA TGG CGA TGT GAA TGG GCA C-3′ and 5′-TAA
TAC GAC TCA CTA TAG GGA GAC AGC AGG ACC ATG TGG TCT CTC-3′; EcR,
5′-TAA TAC GAC TCA CTA TAG GGA GAA AGA ATT CGG TAC CAG GAT
GGC TAT GAG-3′ and 5′-TAA TAC GAC TCA CTA TAG GGA GAT TAG ATC
TCC TCG AGG AAC TTG-3′; E74, 5′-TAA TAC GAC TCA CTA TAG GGA GAT
3
GTC AAG CAA TCG ACA AGT CCG-3′ and 5′-TAA TAC GAC TCA CTA TAG
GGA GAT CGG TGC GTT GAA GTA GGA CG-3′; and BR-C, 5′-GAA TTA ATA
CGA CTC ACT ATA GGG AGA GAT CCA CAG CCA GAA CCA GAC AC-3′ and
5′-GAA TTA ATA CGA CTC ACT ATA GGG AGA AGA CGC TTA AAG GCG
TGG GAG-3′.
Luciferase assay
S2 cells were transfected with plasmids by the DDAB method (Han 1996). In brief, S2
cells were mixed with DDAB, 100 ng firefly luciferase reporter, and 100 ng of renilla
luciferase reporter (pAC5.1-RL) for a total of 1.5 mL insect media on a 12-well plate.
Twelve hours post-transfection, 20 μM 20E or an equal volume of EtOH was added.
Thirty-one hours later, a dual-luciferase assay (Promega) was performed. To carry out
luciferase assays for the mutated reporter, S2 cells were transfected with reporters using
Effectene reagent (Qiagen). Twenty-four hours post-transfection, 10 μM 20E or EtOH
was added. Forty-eight hours later, dual-luciferase assays (Promega) were performed.
Supplementary References
Han, K. 1996. An efficient DDAB-mediated transfection of Drosophila S2 cells.
Nucleic Acids Res 24(21): 4362-4363.
Hyun, S., Lee, J.H., Jin, H., Nam, J., Namkoong, B., Lee, G., Chung, J., and Kim, V.N.
2009. Conserved MicroRNA miR-8/miR-200 and its target USH/FOG2 control
growth by regulating PI3K. Cell 139(6): 1096-1108.
4