Extended Data Fig. 2: The formation of small condensates of HSF1 in the nucleoplasm in response to HS.
(a) Voronoï-based segmentation of HSF1 condensates. Zoomed images were displayed at the bottom. Each molecule has a polygon defined by its neighbouring molecules. Edges of Voronoï polygons are located equidistant from the nearest two molecules. When a new molecule is added, this bisector is cut by the bisectors computed between the old molecules and the new ones. Each new molecule was computed to plot the Voronoï diagram repeatedly until all the molecules were counted. Then a threshold was set twice the average localization density to choose the clusters of HSF1 molecules. (b,c) Representative fluorescence images of widefield and STORM showing the formation of small condensates in different cell lines under NHS (b) and HS (42 °C, 0.5 h) (c) conditions. Scale bars, 2 μm. Zoomed scale bars, 100 nm. (d-f) Cluster analysis of HSF1 in different cell lines under NHS and HS (42 °C, 0.5 h) conditions. Localization per cluster (d), cluster number per cell (e), and cluster size (f) were shown. Data are presented as mean ± s.d. Individual data points correspond to the average value for one cell. n = 19 cells for A549-HS, 27 cells for HEK293T-HS, 18 cells for MDA-MB-231-HS, 15 cells for U2OS-HS, 15 cells for A549-NHS, 12 cells for HEK293T-NHS, 15 cells for MDA-MB-231-NHS, 20 cells for U2OS-NHS pooled from 3 independent experiments. The paired two-tailed Student’s t-test was used to compare the data (e). Boxplots: 25th to 75th percentiles, median, 1.5×interquartile as whiskers (d). (g) Schematic of the single molecule tracking experiment. Using the HaloTag knock-in cells, HSF1 was labelled with JF549 and tracked using HILO illumination. Then the trajectory of each molecule was extracted and analysed. (h) The single molecule trajectory of WT HSF1 and LLPS-incompetent M3 under NHS and HS (42 °C, 0.5 h) conditions. The trajectories were colour-coded according to their diffusion coefficients. n = 10 cells for WT HSF1 under NHS, 10 cells for WT HSF1 under HS, 20 cells for M3 under NHS, 16 cells for M3 under HS pooled from 3 independent experiments. (i) The mean square displacement of HSF1 and LLPS-incompetent M3 under NHS and HS (42 °C, 0.5 h) conditions. (j) The distribution of diffusion coefficient of HSF1 and LLPS-incompetent M3 under NHS and HS (42 °C, 0.5 h) conditions. Images are representative of three independent experiments (b-c).
