Figure 3.

Twinkle and mtSSB depletion have distinct RNA granule phenotypes-1. U2OS cells were treated for 3 days with siRNAs for mtSSB, Twinkle, GRSF1, mtSSB+GRSF1, Twinkle+GRSF1 as well as universal control siRNAs, subsequently incubated for one hour with 2.5 mM BrU, briefly washed, fixed, lysed and incubated sequentially with a BrU, an mtSSB and a DNA antibody for immunofluorescent detection. Shown here are 20 × 30 μm representative details and including not only the individual antibodies but also the merged channel images, as indicated. Full images of the individual antibody detections are shown in Supplementary Figure S1. In addition, we show segmented high contrast images of the dual-channel overlay images of this Figure as a Supplementary Figure S7, having used the ImageJ Squassh plug-in ((13), see also M&M). This and subsequent Figures 4 and 5, for each figure, show parallel treated samples from a single six-well plate. The three knockdown experiments represented here in Figures 3–5 are thus biological repeats, albeit probed with different antibodies. For each individual Figure/experiment, all images were acquired with identical microscope settings, such as LED intensity, exposure time etc, and images were further processed identically. (See also Supplementary Figure S1 for full fields of view).