nHOTAIRM1 recruitment in SG followed with Pepper fRNA. (A) Representative HEK 293T cell expressing both nHOTAIRM1 labeled with HBC620 (magenta) and GFP–G3BP1 (green). Time 00:00:00 corresponds to 0.5 mM NaAsO2 administration (upper row), whereas in the following time points (second and third row) formation of SG can be followed (duration 1 h; interval 1–3 min). All scale bars correspond to 5 μm. (B) Magnification view of yellow box in A showing different nHOTAIRM1 particles and G3BP1 undergoing LLPS throughout the oxidative stress event (yellow and light blue asterisks) and merging (time points 00:39:00 and 00:44:12). Second and fourth row show binary masks of the selected time points highlighting fusion events. Scale bars, 5 μm. (C) Average Manders’ coefficient between nHOTAIRM1 and G3BP1 over time upon NaAsO2 administration. Dots represent average values of Manders’ coefficient. Error bars represent ± SEM. Average and SEM values were calculated from 5 to 10 time-lapse measurements (duration 1 h; interval 1–5 min) collected from three independent biological replicates. Each color (magenta, gray, and green) corresponds to a single biological replicate. (***) P ≤ 0.001 corresponds to two-tailed paired Student's t-test (N = 3). (D) Scatter plot showing intersection area over time calculated from intersection mask between G3BP1 and nHOTAIRM1 signals of A. A polynomial curve was fitted to the points in order to show the increase over time (R2 = 0.8838).
