Supplementary Figure 1
Miyoshi et al

DAPIMerge

myc-Loqs-PD

myc-Loqs-PC

myc-Loqs-PB

myc-Loqs-PA

anti-myc

Supplementary Figure 1
Immunofluorescence analyses using an anti-myc antibody revealed that myctagged Loqs-PC expressed in S2 cells localized in large foci in the cytoplasm
while other isoforms localized evenly in the cytosol. Immunofluorescence
was performed by fixing S2 cells with 2% formaldehyde for 15 min. Cells
were permeabilized using 0.1% Triton X-100. To visualize myc-tagged Loqs
proteins, 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 containing
DAPI (Invitrogen). All images were collected using a Zeiss LSM510 laser
scanning microscope.

Supplementary Figure 2
Miyoshi et al
Loqs-RALoqs-RB
Loqs-RC
Loqs-RDA
ds-ALLds-RB+RC
ds-RC
ds-RC+RD
ds-RD

D

ds-RC+RD

ds-RB+RC

ds-RD

ds-RC

-ds-RD

ds-RC+RD

ds-RC

ds-RB+RC

ds-ALL

ds-EGFP-dsRNA

ds-ALLdsRNAds-EGFP

B- pre-bantam- Loqs-PB- Loqs-PA- Loqs-PC/PD- bantam- pre-miR-8

Cds-RC+RD

ds-RB+RC

ds-RDds-RCds-ALL

ds-EGFP-dsRNA- miR-8- pre-miR-2b- esiRNA-sl-1- CG18854A- miR-2b- esiRNA-1731- U6 snRNA- U6 snRNA

Supplementary Figure 2Loqs-PD depletion causes a severe defect in esiRNA production in S2 cells. (A) A schematic drawing
showing 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 isoform
depletion. ds-EGFP was employed as a negative control. (C) Northern blot analyses using DNA oligo
probes specific for bantam miRNA, miR-8 and miR-2b show that depletion of Loqs-PA and Loqs-PB
caused 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-derived
esiRNA (esiRNA-1731) was also affected by Loqs-PD RNAi. To detect esiRNA-1731 derived from a
retrotransposon, we carried out 1-ethyl-3-[3-dimethylaminopropyl]carbodimide hydrochloride crosslinking reactions for 1 h at 60°C (Pall GS, Hamilton AJ. 2008. Nat Protoc 3: 1077-1084). Probes
used for detecting esiRNA-1731 were as follows; esiRNA-1731#1, 5’-AAG GTG TCG TCG CTG
GTC 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 GTT3’; 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’.

kDa58 -ovarytestis

BS2A- Loqs-PB- Loqs-PA- Loqs-PC/PDRelative mRNA levels

Supplementary Figure 3
Miyoshi et al46 -

3.0S2

2.5
2.0
1.5
1.0
0.50

Relative mRNA levels

Loqs-PA Loqs-PB Loqs-PC Loqs-PD
1.2testis

1.0
0.8
0.6
0.4
0.20

Relative mRNA levels

Loqs-PA Loqs-PB Loqs-PC Loqs-PD
1816141210864
20ovary

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

Supplementary Figure 3
The 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 expression
from 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 cDNAs
were 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 CCA
TCG 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 GAG
TTG AA-3’ for Loqs-RC; 5’-TGC AGG AGA CTC CCA TCG ATT CG-3’ and 5’-TGC
TCG TAA CGA TCG ATG TCT TGA ATG-3’ for Loqs-RD.