Wild type (A, E, F), gfp::sas-5 (B), gfp::sas-5 L141E (C), and gfp::sas-5 I247E (D) worms were treated with sas-5 RNAi (A–D) or simultaneous gfp and sas-5 RNAi (E, F), and the resulting embryos imaged by dual DIC and fluorescence time-lapse microscopy. Top panels: one-cell stage embryos, bottom panels: same embryo ∼25 min later. GFP images are maximum intensity projections of 8 z-sections taken at 0.5 μm intervals. Time from the first image is shown in mm:ss. Insets in panels B, C, E and F show 2.5 fold enlargements of boxed regions. (G) Quantification of GFP fluorescence intensity in live one-cell stage embryos, shown as a percentage of the average value for wild-type GFP-SAS-5. Rescue of endogenous sas-5 depletion upon dual RNAi treatment depended on the level of exogenous GFP-SAS-5 expression. Importantly, note that levels of GFP-SAS-5 below those of either of the GFP-SAS-5 mutants, reduced using gfp RNAi, were able to rescue the sas-5 (RNAi) phenotype, indicating that the lack of rescue in the latter two strains is not due to insufficient expression levels. To rule out the possibility of sas-5 (RNAi) rescue being due to dilution of the sas-5 RNAi with gfp RNAi, embryos expressing GFP-SAS-5 L141E were scored under the same conditions, with all embryos showing the sas-5 (RNAi) phenotype (data not shown) Furthermore, GFP-SAS-5 levels at only ∼5% of the levels in the sas-5 (RNAi) condition, failed to rescue, further indicating that sas-5 (RNAi) is functional under these conditions. (H) Western blots of 50 adult worms from each indicated strain probed with antibodies against SAS-5 or GFP, as indicated, and against α-tubulin as loading control. Molecular weight markers are indicated as black dashes. Endogenous SAS-5 exists as two isoforms: the long 404 amino acid isoform and a shorter 288 amino acid isoform, which is likely the result of trans-splicing (Wormbase, F35B12.5c). Note that exogenous gfp::sas-5 results in the expression of the longer isoform only.