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Files in this Data Supplement:
Fig. S1. RAB-11.1 and SYN-4 are required for the proper eggshell formation and cytokinesis. (A-C) RNAi of rab-11.1 or syn-4 causes osmotic sensitivity of embryos. The embryos are dissected from mock (A), rab-11.1(RNAi) (B) and syn-4(RNAi) (C) animals, incubated in 300 mM KCl solution and observed. (D,E) RNAi of rab-11.1 or syn-4 results in multinucleated embryos without cytokinesis. The nuclei of mock (A), rab-11.1(RNAi) (B) and syn-4(RNAi) (C) embryos were observed by using GFP::histone H2B. Scale bars: 10 µm.
Fig. S2. Knockout of syn-4 causes a defect in gonadogenesis. Gonads were dissected from wild-type (A-D) or syn-4; T01B11.4(ok372; E-H) animals and stained with WGA-TRITC. Images of Nomarski (A,C,E,G) and WGA staining (B,D,F,H) are shown. In syn-4; T01B11.4(ok372), gonads are severely disorganized and no distinct oocytes and embryos are produced. Spermatogenesis is also affected by syn-4; T01B11.4(ok372). This deletion allele (ok372) lacks a part of syn-4 and T01B11.4, the flanking gene of syn-4. Because a deletion of T01B11.4 alone (gk300) is superficially wild type and does not cause such a sterile phenotype, the phenotypes of ok372 observed in the germline are likely to be due to the deletion of syn-4. Scale bars: 20 µm. SP, spermatheca.
Fig. S3. mCherry::SNB-1 colocalizes with CAV-1::GFP on cortical granules in oocytes. mCherry::SNB-1 and CAV-1::GFP were co-expressed in the germline and subcellular localization was observed in oocytes. The insets show 2× enlargements of the boxed areas. Scale bar: 10 µm.
Fig. S4. Cortical granule exocytosis is not impaired in snb-1(md247) or snb-1(RNAi) animals. Embryos of snb-1(md247) expressing GFP::CAV-1 (A,A′) or snb-1(RNAi) expressing CAV-1::GFP (B,B′) were observed. Images of GFP fusions with CAV-1 (A,B) and Nomarski (A′,B′) are shown. Cortical granule exocytosis and subsequent degradation of GFP fusions in the early embryos are not impaired under these conditions. Scale bar: 10 µm. SP, spermatheca. (C) Efficiency of RNAi-mediated depletion of SNB-1 in the germline. Animals expressing GFP::SNB-1 in the germline were treated with RNAi of snb-1, and the level of GFP::SNB-1 was examined by western blotting using anti-GFP antibody. Asterisk indicates a non-specific band.
Fig. S4. Cortical granule exocytosis is not impaired in snb-1(md247) or snb-1(RNAi) animals. Embryos of snb-1(md247) expressing GFP::CAV-1 (A,A′) or snb-1(RNAi) expressing CAV-1::GFP (B,B′) were observed. Images of GFP fusions with CAV-1 (A,B) and Nomarski (A′,B′) are shown. Cortical granule exocytosis and subsequent degradation of GFP fusions in the early embryos are not impaired under these conditions. Scale bar: 10 µm. SP, spermatheca. (C) Efficiency of RNAi-mediated depletion of SNB-1 in the germline. Animals expressing GFP::SNB-1 in the germline were treated with RNAi of snb-1, and the level of GFP::SNB-1 was examined by western blotting using anti-GFP antibody. Asterisk indicates a non-specific band.
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