Supplementary Figure 6: Effect of flavopiridol treatment on RNA Pol II dynamics.
From: RNA polymerase II clustering through carboxy-terminal domain phase separation
(a) Cumulative distribution functions (CDF) for displacements. The CDF of displacements for the representative time-lags Δτ = 22.5 ms and Δτ = 30.0 ms are shown for Halo-RPB1-25R, Halo-RPB1-52R and Halo-RPB1-70R after treatment with flavopiridol. The data shown is merged from three independent replicates. (b) Model fit to displacement histograms. Raw displacements from spaSPT data for six different time-lags are shown for Halo-RPB1-25R, Halo-RPB1-52R and Halo-RPB1-70R. Model-fitting from a two-state (bound vs. free) model is overlaid, from which the diffusion constants and subpopulation sizes were calculated. The data shown is merged from three independent replicates (n = 13, 15, 28 cells in total for Halo-RPB1-25R, -52R, -70R, respectively). (c) Bound fractions of Halo-RPB1-25R, -52R and -70R after flavopiridol treatment. The bound fraction was inferred from two-state model-fitting to the spaSPT displacement data using Spot-On (Hansen, A.S. et al., Elife 7, 2018). Each of three independent replicates was fitted separately and bar graphs show the mean and standard error. (d) Diffusion coefficients of the free population of Halo-RPB1-25R, -52R and -70R. Free diffusion coefficients were inferred from two-state model-fitting to the spaSPT displacement data using Spot-On (Hansen, A.S. et al., Elife 7, 2018). Each of three independent replicates was fitted separately and bar graphs show the mean and standard error. (e) FRAP data of Halo-RPB1-25R (red), Halo-RPB1-52R (blue) and Halo-RPB1-70R (grey) after flavopiridol treatment. The data were fitted to a reaction dominant two-state model (Sprague, B.L. et al., Biophys J. 86, 3473-95, 2004; Hansen, A.S. et al., Elife 6, 2017). FRAP curves represent the mean across n = 5 independent measurements for each cell line and error bars show the standard error. We performed 50 iterations using 50% of the data in each to estimate the error (standard deviation of the subsampling) on the bound fraction. For comparison purposes, the FRAP curves of untreated cells (presented in Fig. 5f) are overlaid (light grey).
