Figure 9.

Both GRSF1 and mtSSB depletion result in dsRNA accumulation. U2OS cells were treated for 3 days with siRNAs for mtSSB, Twinkle, GRSF1, mtSSB+GRSF1, mtSSB+Twinkle as well as universal control siRNAs, and were sequentially processed and incubated with the J2 dsRNA (green), mtSSB or GRSF1 (red), and a DNA antibody (not shown) for immunofluorescent detection. Shown are the J2, and mtSSB or GRSF1 (as indicated) antibody incubations and their merged images. For all the targeted siRNAs, images were chosen in which also cells with failed knockdown are visible, as judged from the mtSSB/GRSF1 antibody incubations. These cells are indicated with either (–) for a failed knockdown or (+/–) to indicate either a partial knockdown or a failed double knockdown where at least one of the proteins was not depleted. For the Twinkle knockdown, the GRSF1 antibody incubation was used as a diagnostic antibody to determine knockdown, showing many more and more intense RNA granule-GRSF1 foci in those cells where the knockdown did not work properly. A similar experiment, but using the RNA:DNA hybrid antibody S9.6, is shown in Supplementary Figure S6.