Figure EV1. Loss of Zuc and Gasz impacts the cellular localization of Armi and Piwi in OSCs.

1. Western blotting using anti‐Gasz monoclonal antibody shows that the Gasz signal disappeared upon Gasz RNAi treatment in OSCs. Anti‐Gasz monoclonal antibody was produced in this study. β‐Tubulin (β‐Tub) was detected as a loading control.
2. Western blotting shows that the levels of Armi and Piwi in the cytoplasmic fraction after mitochondrial isolation (cyto) were little impacted by Zuc depletion, while the level of Armi in the mitochondrial fraction (mito) increased.
3. Larger immunostaining views of the cell images shown in Fig 1C and D. The cells appeared in the main figures are boxed with white dotted line. In “Zuc KD” panel, the upper cell appeared in Fig 1C, while the lower one appeared in Fig 1D. Armi behaved similarly in the cells other than the one shown in Fig 1C and D. The scale bar represents 5 μm.
4. Upper: Localization of Piwi (green) in OSCs upon Zuc and Gasz depletion. Yb bodies are shown in red. Lower: Larger immunostaining views of the cell images shown in upper panels. The cells appeared in the upper panels are boxed with white dotted line. Piwi was mislocalized in the cytosol upon the loss of Zuc and/or Gasz. Some fraction of Piwi was detected at Yb bodies as has previously been reported 24. The scale bar represents 5 μm. DAPI (blue) shows the nuclei.
5. Left: Localization of Armi (green) in OSCs upon Gasz depletion. Mitochondria are shown in red. Right: Larger immunostaining views of the cell images shown on the left. The scale bar represents 5 μm. DAPI (blue) shows the nuclei.
6. Piwi localizes to Yb bodies after Armi has localized there. Upon Piwi–piRISC formation by Zuc on the outer membrane of mitochondria, Armi goes back to Yb bodies, while Piwi–piRISC is translocated to the nucleus for silencing. The details and requirements of inter‐organelle translocation of Armi and Piwi–pre‐piRISC remain unknown (red dotted line with an arrowhead).