(
A) Western blots showing CG2678 or Rhino protein levels in ovarian lysate from indicated genotypes (Ponceau staining served as loading control). Changes in protein levels and isoform ratios between control and
rhino depleted ovaries were not reproducible and are likely caused by differences in ovary morphology, which distort the protein composition across samples. (
B) Confocal images showing immunofluorescence signal for CG2678 and Rhino in nurse cells of MTD-Gal4-driven control or
CG2678 knockdown ovaries (scale bar: 5 µm). (
C) Confocal images showing FLAG-tagged CG2678 and Rhino signal in nurse cells expressing an internally FLAG-tagged CG2678 in the CG2678 mutant background (see
Figure 2B for location of FLAG tag; scale bar: 5 µm). (
D) Confocal images showing the localization of GFP-Rhino with H3K9me3 (top), Deadlock (middle), or Nxf3 (bottom) in
CG2678 null mutant nurse cells (scale bar: 5 µm). (
E) Scatter plot of genomic 1-kb tiles contrasting average log2-fold enrichment for Rhino ChIP-seq signal in
CG2678 knock out versus
w1118 control ovaries (three and two replicate experiments, respectively). (
F) Violin plots showing average log2-fold H3K9me2 ChIP-seq enrichment for Rhino-bound 1-kb tiles in MTD-Gal4 background in heterochromatin (HC) and along chromatin arms (EC) contrasting control (n=2) to RNAi-mediated
CG2678/kipferl knockdown (n=1). Rhino-dependent piRNA clusters
38C,
42AB, and
80F are shown separately. Box plots show median (center line), with interquartile range (box) and whiskers indicate 1.5x interquartile range. (
G) Confocal images showing the localization of piRNA cluster RNA via RNA FISH in respect to GFP-Rhino in nurse cells of
w1118 or
CG2678/kipferl locus deletion flies (scale bar: 5 µm). Imaging conditions were chosen to avoid photo bleaching and to optimally display the large Rhino-GFP accumulations to show that these are not corresponding to either of these clusters. (
H) Confocal images showing
Rsp and
1.688 Satellite RNA FISH signal in respect to GFP-Rhino in nurse cells of
CG2678/kipferl,nxf3 double mutant flies (scale bar: 5 µm).