Fig. 9: Organisation and dynamics of nuclear NDP52 following transcription inhibition.
From: Autophagy receptor NDP52 alters DNA conformation to modulate RNA polymerase II transcription
A Example of STORM rendering and cluster map, generated following DBSCAN analysis of RNAPII-pSer5 following α-amanitin treatment. Scale bar = 5 μm. B Calculated number of RNAPII-pSer5 molecules in the nucleus of non-treated vs. α-amanitin-treated cells (n = 10 cells). C Percentage of RNAPII-pSer5 molecules in clusters for non-treated vs. α-amanitin-treated cells (n = 10 cells). D Number of clusters in selected ROIs for RNAPII-pSer5 in non-treated vs. α-amanitin-treated cells. n = 10 cells (non-treated) n = 10 cells (α-amanitin). E Confocal image of NDP52 in HeLa cells following treatment with transcription inhibitor α-amanitin. Scale bar = 5 μm. Hoechst DNA stain is shown in blue. F Example STORM image of NDP52 in HeLa cells treated with α-amanitin and corresponding cluster map. Scale bar = 5 μm. Clustered molecules are shown in green. G Linearised Ripley’s K Function, L(r)−r, calculated for selected ROIs from STORM images. Ripley’s K values red = α-amanitin-treated cells and blue = non-treated cells (values are the same as shown in Fig. 2C). n = 11 (α-amanitin) n = 12 (non-treated). H Percentage of molecules in clusters in non-treated HeLa cells compared to α-amanitin treatment. Values for non-treated cells are the same as shown in Fig. 2G. n = 11 (α-amanitin) n = 12 (non-treated). I Histogram of diffusion constants for Halo-NDP52 non-treated (blue) and α-amanitin-treated (red) HeLa cells. Dotted lines represent the applied threshold to differentiate between static and dynamic molecules (14,322 molecules from 51 cells for non-treated condition and 14,492 molecules from 50 cells). Non-treated cell values are the same as shown in Fig. 3G.J Percentage of molecules considered static (D < 0.1 μm2/s), slow moving (0.1 < D < 1 μm2/s) or diffuse (D > 1 μm2/s) per cell. n = 51 cells. (non-treated—same as Fig. 3J) and n = 50 (α-amanitin). K Diffusion coefficient values and (L) Anomalous diffusion constants (α) for Halo-NDP52. Each data point represents the mean value for a cell. n = 51 (non-treated—same as Fig. 3H) and n = 50 (α-amanitin). For all experiments: errors bars are mean ± SEM. ns = p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 by a two-tailed t-test. Raw data are available in the Source Data file.
