Live-cell imaging of circular and long noncoding RNAs associated with FUS pathological aggregates by Pepper fluorescent RNA

nHOTAIRM1 recruitment in ALS-like granules followed with Pepper fRNA. (A) Representative cell expressing both nHOTAIRM1 labeled with HBC620 (magenta) and GFP-FUS^P525L (green). Time 00:00:00 corresponds to 0.5 mM NaAsO₂ administration (upper row), whereas in following time points (second and third row) formation of FUS^P525L condensates can be followed (duration 1 h; interval 1–5 min). Scale bars, 5 μm. (B) Magnification view of yellow box in D showing nHOTAIRM1 and FUS^P525L undergoing LLPS throughout the oxidative stress event (yellow asterisk) and finally merging (time point 00:38:51). 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 FUS^P525L 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 FUSP^525L and nHOTAIRM1 signals of Figure 6A. A polynomial curve was fitted to the points in order to show the increase over time (R² = 0.9035).