S2R+ cell transfection

100 μl of cells at 10⁶ cells/ml resuspended in Schneider's Drosophila medium (GIBCO, Invitrogen) were plated in 96-well plates. Cells were transfected with dsRNA or co-transfected with dsRNA and the corresponding sensor using Effectene (QIAGEN) following the manufacturer's instructions. Thirty minutes after transfection 50 μl Schneider's Drosophila medium (GIBCO, Invitrogen), completed with 10% heat-inactivated fetal calf serum, 100 U/ml penicillin and 100 mg/ml streptomycin were added. Cells were grown at 23°C without CO₂. After 24–48 h, CuSO₄ was added to a final concentration of 600 μM and GFP fluorescence was followed using an inverted epifluorescence basic microscope. For Ago2-mediated miRNA pathway involvement (automiG), cells were co-transfected with 0.1 μg of automiG-vector and 0.32 μg of dsRNA targeting either Ago2, CG7009 or Ftz, Dcr1, Dcr2, Drosha, Ago1. Forty eight hours later, the automiG promoter was induced by adding CuSO₄ to a final concentration of 600 μM (more details in (66)).

For the luciferase assay experiment, S2R+ cells were treated for 4 days with dsRNA inactivating specifically the indicated genes. Cells were co-transfected with two plasmids expressing the Firefly and Renilla luciferases in addition to a dsRNA against Firefly. Luciferases activities were measured 48 h after transfection. The averages of the activity ratios from Firefly/Renilla luciferases from three independent biological replicates were plotted normalized to the average of a control dsRNA (GFP) which was set to 1 (+/– the standard deviations). * indicates P < 0.05 in a Student's t-test.