Supplementary Figure 1. The level of pri-mir-8 gradually decreases while those of BR-C and E74 increase during 3rd instar larval development.

Transcription

1 qrt-pcr RT-PCR Relative pri-mir-8 level Early 3rd (72h) Mid 3rd (96h) Late 3rd (119h) BR-C E74 mtl rrna Early 3rd (72h) Mid 3rd (96h) Late 3rd (119h) 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.

2 Northern Blot 3h 6h 16h 24h 48h 72h mir-8 mir-8/ 2S rrna S rrna Supplementary Figure 2. Treatment of 20-hydroxyecdysone () in S2 cells progressively decreases the mir-8 levels analyzed by northen blot. Ethanol () is a vehicle for. 2S rrna is a loading control used for mir-8 quantification. Times post / treatment are denoted above the blot.

3 position (strand) sequence factor name (+) aaaaacaggaaaagggt E74A (+) ttaaacgggaaagacaa E74A (+) acgggaaagacaagaata BR-C Z (+) taattatatacaaacaca BR-C Z (-) ttatttttactat BR-C Z (-) tttttttttttaa BR-C Z (-) tttattttaaaaa BR-C Z (+) aatagaaataaaa BR-C Z (+) taaataaaccaaa BR-C Z (+) aaattataggcaattaag BR-C Z1-718 (+) tttttattaccaaattcg BR-C Z1-632 (+) taaccatgaacaagtctt BR-C Z1-456 (+) ccaaaaaggaagccgtg E74A -402 (+) agttaaaaatcaaatcac BR-C Z1 113 (+) atagtaaaataaa BR-C Z4 128 (+) atagaaaacagaa BR-C Z4 418 (+) tcaataaactaat BR-C Z4 437 (-) tggctttgtttttgtttta BR-C Z1 516 (-) ttaaatttacaaa BR-C Z4 539 (+) aagcgacaaacaaaattc BR-C Z1 557 (+) aaaagaaagaaaa 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.

4 Cloned gdna PR1-miR PR2-miR-8 TF binding site: BR-C E74 CG34460 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: ].

5 Luciferase Assay Relative expression (FL/RL) pgl3 null p=0.01 p=0.03 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 on reporter activity (n=4). p values are denoted for the significance. Error bars denote SEM.

6 Timing of Puparium Formation armg4/ /- 6.0 arm>ecr i /- 8.2 cgg4/ /- 7.2 cg>ecrdn / AEL (hr) 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.

7 1.5 qrt-pcr Relative dmyc level cgg4/+ cg>ecrdn 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.

8 Pupal Volume p= p=0.01 Relative size ushg4/+ ush>ush ush>ecr i ush>ecr i & ush 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.

9 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 TAA- 3 ; 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- 32 ATP] 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

10 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 3 rd 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, 1 st 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 3 rd 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 MgCl 2, 0.2 mm EDTA, 1 mm NaVO 4, 50 mm NaF, 1 mm DTT, and 25% glycerol) as previously described (Hyun et al. 2009). Immobilon TM PVDF 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

11 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 or with an equal volume of 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

12 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 or an equal volume of 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 or was added. Forty-eight hours later, dual-luciferase assays (Promega) were performed. Supplementary References Han, K An efficient DDAB-mediated transfection of Drosophila S2 cells. Nucleic Acids Res 24(21): Hyun, S., Lee, J.H., Jin, H., Nam, J., Namkoong, B., Lee, G., Chung, J., and Kim, V.N Conserved MicroRNA mir-8/mir-200 and its target USH/FOG2 control growth by regulating PI3K. Cell 139(6):