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Fig. S1. Specificity of the anti-PAPI antibody. (A) HA-tagged full-length PAPI and a series of PAPI deletion constructs were expressed in yeast cells. The cell lysates were probed with anti-HA and anti-PAPI antibodies. The empty vector that only encodes HA tag was used as a negative control. The anti-PAPI antibody recognizes full-length PAPI and all the PAPI deletions, but not the negative control. (B) A western blot of ovarian lysates from wild-type flies with anti-PAPI antibody.
Fig. S2. PAPI associates with PIWI proteins in vivo. Endogenous PAPI was immunoprecipitated from embryonic extract using anti-PAPI antibody and subject to western blotting analysis. The immunoblot was probed with indicated antibodies. Extract plus beads (−Ab) and antibody plus beads (−Extract) were used as negative controls for immunoprecipitation. IgG was probed as a loading control.
Fig. S3. PAPI binds to the sDMAs in the N terminus of PIWI via its TUDOR domain. (A,B) Y2H assay indicating that the N-terminal domain of PIWI interacts with the PAPI-FL and PAPI-CT. The B42AD moiety of the prey plasmid pJG4-5 was used as a negative control. The positions of amino acid residues at various domain junctions are indicated. (C,D) Y2H assay indicating that the C-terminal domain of PAPI interacts with PIWI. Drosophila Bicoid protein and LexA were used as negative controls. The positions of amino acid residues at various domain junctions are indicated. (E) The sDMAs in the N-terminal of PIWI are required for PAPI-PIWI interaction. Double sDMA mutant PIWI does not interact with PAPI. Arginine to lysine mutations are indicated.
Fig. S4. Flag-Myc-PAPI transgene is localized to the nuage. (A) Flag-Myc-tagged PAPI (FM-PAPI) expression was driven by Act5C-Gal4 driver. Act5C-Gal4/+ and Act5C-Gal4/UAS-FM-PAPI fly lysates were probed with anti-PAPI and anti-Myc antibodies. Endogenous PAPI and the FM-PAPI transgene are indicated. (B) Immunostaining of ovaries from Act5C-Gal4/+ and Act5C-Gal4/UAS-FM-PAPI flies using an anti-Myc antibody. The FM-PAPI protein is enriched in the perinuclear nuage (arrowheads). (C) FM-PAPI colocalizes with PAPI in the nuage (arrowheads) in nurse cells of a stage 4 egg chamber.
Fig. S5. Colocalization of PAPI with PIWI and AUB. (A) Co-immunostaining of PAPI and PIWI in a wild-type egg chamber. PAPI is mainly in the cytoplasm and enriched in the nuage. PIWI is predominantly in the nucleus. (B) Co-immunostaining of PAPI and AUB in a wild-type egg chamber. AUB and PAPI partially colocalize in the nuage.
Fig. S6. Knockdown of PAPI by RNAi affects female fertility. (A) Quantitative RT-PCR showing the transcript levels of papi, relative to rp49, from wild-type (WT), Act5C-Gal4/+, UAS-papi-RNAi/+ and Act5C-Gal/UAS-papi-RNAi flies. Transcript levels from wild-type ovaries were set as 1, and fold changes are indicated. (B) Western blot of PAPI and GAPDH from Act5C-Gal4/+ and Act5C-Gal/UAS-papi-RNAi flies. PAPI protein levels were normalized to GAPDH, and fold-changes are indicated. (C) papi RNAi flies lay fewer eggs. Eggs laid by Act5C-Gal4/+, UAS-papi-RNAi/+ and Act5C-Gal/UAS-papi-RNAi female flies were counted from day 0 to day 5.
Fig. S7. Target specificity of papi knockdown via RNAi. (A) Blast search was performed to identify the potential off targets of papi RNAi. The four top score hits of papi shRNA in the fly genome are shown as short black bars underneath the papi shRNA sequence. Among them, two are in RNA-coding genes: CG11160 and CG31935. (B) Sequence alignment of papi shRNA with potential off targets. (C) Quantitative RT-PCR was performed to determine the expression levels of the three mRNAs from CG11160 and CG31935 that contain target sequences of papi shRNA, relative to rp49, in Act5C-Gal4/+ and Act5C-Gal4/papi-RNAi flies. Transcript levels from Act5C-Gal4/+ ovaries were set as 1 and fold changes are indicated.
Fig. S8. The nuage is not disrupted in papi-deficient ovaries. Immunostaining of PIWI (A), AUB (B), and VASA (C) in wild-type and Act5C-Gal4/papi-RNAi egg chambers. The localization of PIWI, AUB and VASA are not affected in papi-deficient ovaries, indicating that the nuage is not disrupted in these ovaries.
Fig. S9. Germline PAPI is required for AGO3 localization to the nuage. (A) Immunostaining of PAPI in wild-type and nosVP16-Gal4/papi-RNAi egg chambers. Expression of papi-RNAi under the nosVP16-Gal4 driver specifically reduced PAPI in germline cells but not somatic follicle cells. (B) Immunostaining of PAPI in wild-type and nosVP16-Gal4/papi-RNAi egg chambers. AGO3 is delocalized from the nuage in nosVP16-Gal4/papi-RNAi egg chambers.
