anti-myc DAPI Merge

myc

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yc-L

oqs-

PB

myc

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oqs-

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Supplementary Figure 1Miyoshi et al

Supplementary Figure 1Immunofluorescence analyses using an anti-myc antibody revealed that myc-tagged Loqs-PC expressed in S2 cells localized in large foci in the cytoplasmwhile other isoforms localized evenly in the cytosol. Immunofluorescencewas performed by fixing S2 cells with 2% formaldehyde for 15 min. Cellswere permeabilized using 0.1% Triton X-100. To visualize myc-tagged Loqsproteins, cells were stained with a monoclonal anti-c-myc antibody (9E10).Alexa Fluor 546-tagged anti-mouse IgG was used as a secondary antibody.Cells were also mounted with SlowFade Gold Antifade Reagents containingDAPI (Invitrogen). All images were collected using a Zeiss LSM510 laserscanning microscope.

Loqs-RALoqs-RBLoqs-RCLoqs-RD

ds-ALL

ds-RC

ds-RD

ds-RB+RC

ds-RC+RD

A

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- ds-E

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LL

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dsRNA

- pre-bantam

- bantam

- miR-8

- miR-2b

- pre-miR-8

- pre-miR-2b

- U6 snRNA

- esiRNA-sl-1

- CG18854A

- esiRNA-1731

- U6 snRNA

- ds-E

GF

P

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LL

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dsRNA

- ds-E

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ds-R

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RC

ds-R

C

ds-R

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RD

ds-R

D

dsRNA

- Loqs-PB

- Loqs-PA- Loqs-PC/PD

C

Supplementary Figure 2Miyoshi et al

Supplementary Figure 2Loqs-PD depletion causes a severe defect in esiRNA production in S2 cells. (A) A schematic drawingshowing the regions in the Loqs coding gene to which the dsRNAs for RNAi, ds-ALL, ds-RB+RC,ds-RC, ds-RC+RD, and ds-RD correspond. (B) Western blot analysis using anti-Loqs antibodies onS2 cell lysates after RNAi treatment shows the efficiency and the specificity of Loqs isoformdepletion. ds-EGFP was employed as a negative control. (C) Northern blot analyses using DNA oligoprobes specific for bantam miRNA, miR-8 and miR-2b show that depletion of Loqs-PA and Loqs-PBcaused a severe defect in miRNA biogenesis. A probe for U6 snRNA was used as a loading control.(D) Northern blot analyses using DNA oligo probes specific for esiRNA-sl-1 and CG18854A-esiRNAshow that depletion of Loqs-PD led to a severe defect in esiRNA biogenesis. DM1731-derivedesiRNA (esiRNA-1731) was also affected by Loqs-PD RNAi. To detect esiRNA-1731 derived from aretrotransposon, we carried out 1-ethyl-3-[3-dimethylaminopropyl]carbodimide hydrochloride cross-linking reactions for 1 h at 60°C (Pall GS, Hamilton AJ. 2008. Nat Protoc 3: 1077-1084). Probesused for detecting esiRNA-1731 were as follows; esiRNA-1731#1, 5’-AAG GTG TCG TCG CTGGTC TAC-3’; esiRNA-1731#2, 5’-CCG GTT CAT CAC TCT AGG ACG-3’; esiRNA-1731#3, 5’-GGT CGC CAT CTC GTT GCA CCT-3’; esiRNA-1731#4, 5’-TGC TCT GCT CGT TGA TCG GTT-3’; esiRNA-1731#5, 5’-GGC CAC GAT CAT CTT GAG TGT-3’. Oligo DNAs used as probes were:miR-2b, 5’-GCT CCT CAA AGC TGG CTG TGA TA -3’; miR-8, 5’-GAC ATC TTT ACC TGA CAGTAT TA-3'; esiRNA-18854A, 5'-TCA TTT GAT CCA TAG TTT CCC GT-3’.

Supplementary Figure 3Miyoshi et al

Supplementary Figure 3The expression levels of Loqs isoforms vary among tissues. (A) Western blot analysis wasperformed on S2, testis and ovary lysates using anti-Loqs antibodies. (B) RNA expressionfrom each Loqs isoform was measured by quantitative RT-PCR in S2, testes and ovaries.Total RNAs isolated from S2, testes and ovaries were treated with DNase I and cDNAswere synthesized using Transcriptor Reverse Transcriptase (Roch) and oligo-dT primer.PCR were conducted with LightCycler 480 (Roch) and SYBR Premix Ex Taq (TaKaRa).The primer sequences used are; 5’-TGC AGG AGA CTC CCA TCG ATT CG-3’and TGTTCT TCA AGC AAG TCT TTT CGC CC for Loqs-RA; 5’-TGC AGG AGA CTC CCATCG ATT CG-3’and 5’-ATT TTC ACT ACT GCG GGG TTC GC-3’for Loqs-RB; 5’-TGC AGG AGA CTC CCA TCG ATT CG-3’ and 5’-ACA CAC GCC AAT GGT GAGTTG AA-3’ for Loqs-RC; 5’-TGC AGG AGA CTC CCA TCG ATT CG-3’ and 5’-TGCTCG TAA CGA TCG ATG TCT TGA ATG-3’ for Loqs-RD.

- Loqs-PA

- Loqs-PB

- Loqs-PC/PD

S2

test

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ovar

y

58 -

46 -

kDa

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1.0

1.5

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0

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1.0

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0.4

0.2

0

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12

16

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10

6

2

Loqs-PA Loqs-PB Loqs-PC Loqs-PD

Loqs-PA Loqs-PB Loqs-PC Loqs-PD

Loqs-PA Loqs-PB Loqs-PC Loqs-PD

S2

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