Fig. 4.

Northern blot and hybridization for detection of the primary and secondary siRNAs in flies with the transgene egfp RNAi. A diagram illustrating the probes and target sequence is provided in Fig. S3 and S4. a Detection of the syt anti-sense 2° siRNAs with syt sense probe, compared with control (EGFP.dsRNA + syt.eGFP) without either rrf-1 or ego-1, in which the 2° siRNAs (blue) were produced by endogenous non-canonical RdRp activity, the 2° siRNA production in the presence of RRF-1 (red) was dramatically increased, while siRNA production in the presence of EGO-1 was dramatically reduced and difficult to see with naked eye (yellow), indicating an enhanced transitive RNAi pathway in the presence of RRF-1 but not EGO-1. The detection of syt sense 2° siRNAs in c is consistent with the results shown in a. In comparison, in b, EGFP anti-sense 1° siRNAs without RdRp (blue) and with RRF-1 (red) were detected at a similar signal strength, but production of these siRNAs was dramatically decreased (yellow) when EGO-1 was present, indicating an independent, direct silencing of the EGFP dsRNA hairpin transgene by EGO-1, which is consistent with the EGFP sense 1° siRNAs detection in d. In addition, independent silencing by EGO-1 was achieved not through producing 1° or 2° sense or anti-sense siRNAs (green boxes in all panels)