(A) A Venn diagram showing the impact of the candidates on the siRNA, endo-siRNA and miRNA assays. (B) Candidates were sorted into various categories based on their annotated/verified function. (C) A heat map of the candidates and their scoring patterns in multiple assays. The scores assigned to individual genes in a given assay reflect the relative activity of the pathway upon knockdown of candidate gene expression. Red indicates an increase in silencing and green indicates a decrease. As each candidate is represented by multiple independent dsRNAs, presented are the average scores from all independent dsRNAs targeting a given gene. Scores for individual dsRNAs are shown in Table S1.

Eye phenotypes of the corresponding genotypes are shown.
(A) Ore^R
(B) w¹¹¹⁸
(C) w⁺,GMR-wIR/yw; dcr-2^L811fsX,FRT42D/CyO; EGUF/+
(D) w⁺,GMR-wIR/yw; dcr-2^L811fsX,FRT42D/FRT42D,GMR-hid,Cl; EGUF/+
(E) w⁺,GMR-wIR/yw; EGUF/+; FRT82B/FRT82B,Cl,GMR-hid
(F) w⁺,GMR-wIR/yw; EGUF/+; bel⁶,FRT82B/TM2
(G) w⁺,GMR-wIR/yw; EGUF/+; bel⁶,FRT82B/FRT82B,Cl,GMR-hid
(H) w⁺,GMR-wIR/yw; EGUF/+; hsc70-4^54.1,FRT82B/FRT82B,Cl,GMR-hid

(A) A heat map of steady-state pri-miRNA and miRNA levels upon knockdown of candidates. Steady state levels of CXCR4 and those of endogenous miR-2b upon knockdown of 43 selected candidates were examined by Northern blotting and semi-quantitative RT-PCR (q-PCR). miRNA levels were quantified and normalized first against U6 RNA levels, and then against the average of multiple controls (cells treated with dsRNA against LacZ). Also shown are steady-state levels of pri-CXCR4 measured by q-PCR, and the relative miRNA pathway activities associated with the representative dsRNA. Red indicates an increase in RNA levels, or an increase in the activity of the miRNA pathway, and green indicates a decrease. Presented are the average from 2 to 4 independent Northern blotting experiments and results from individual independent q-PCR assays. The scores associated with each candidate were from experiments involving one representative dsRNA targeting that gene (Table S3). (B) A heat map of endo-siRNA levels upon knockdown of candidates. Steady-state levels of esi-2.1 upon knockdown of 36 selected candidates were examined by q-PCR using multiple independent RNA samples. Quantification was performed as described in A. Also shown are the relative esi-2.1-mediated gene silencing activities. The scores associated with each candidate were from experiments involving one representative dsRNA (Table S4).

(A) Cytoplasmic extract from S2-NP cells was fractionated by size exclusion chromatography, and the fractions were immunoblotted using antibodies against Bel and components of the RNAi pathway, as indicated. The elution profile of molecular markers is shown on top of the panel. (B) FLAG-tagged proteins (as labeled above the panel) were expressed in S2-NP cells and anti-FLAG immunoprecipitates were prepared. The samples were evenly split, and one set were treated with RNase while the other served as control. The samples were immunoblotted sequentially using antibodies against VIG, Ago2, FMR, Ago1 and FLAG. A small amount of cell extract was processed in parallel as input control. Non-specific bands are marked with a “*”. Note that the images shown in lanes 9 through 12 and 23 through 24 of the anti-FLAG panel were from films that were subjected to different exposure times from the rest due to differences in expression levels. (C) Small RNAs are enriched in the Bel and ribosomal protein immunoprecipitates. Expression constructs for FLAG-tagged proteins together with that for the CXCR4 siRNA mimetic were transfected into S2-NP cells. Total RNA was extracted from FLAG immunoprecipitates, and subjected to Northern blotting using probes against Bantam, esi-2.1 and CXCR4. To avoid signal saturation, approximately ~25% of the RNA recovered from the Ago1 and Ago2 samples was loaded compared to the rest of the samples. Total RNAs recovered from 10% of the cell extracts were processed in parallel as input control.