Extended Data Fig. 1: HSF1 undergoes LLPS during HS.
(a) Prediction of intrinsically disordered residues in HSF1 protein by Predictor of Natural Disordered Regions (PONDR). The disordered region is shown by a green horizontal line. The domain organizations of HSF1 are shown at the bottom. (b) Fluorescence images of HeLa cells transfected with HSF1–EGFP under NHS and HS (42 °C, 0.5 h) conditions. Scale bars, 5 μm. (c) Frequency distribution of the number of HSF1 nSBs per cell. 2256 nSBs were analysed in 139 cells pooled across 3 independent experiments. (d) Roundness distribution of the HSF1 nSBs. 2256 nSBs were analysed in 139 cells pooled across 3 independent experiments. (e,f) FRAP recovery of nSBs formed by HSF1. Cells were heat-shocked for 0.5 h at 42 °C to induce phase separation and FRAP was performed after HSF1 droplets emerged. Whole droplets were bleached for the analysis of exchange between nucleoplasm and nSBs, while half droplets were bleached for the analysis of exchange within nSBs and exchange between nucleoplasm and nSBs. Data are presented as mean ± s.d. n = 16 droplets for whole, n = 16 droplets for half collected from 3 independent experiments. Scale bars, 1 μm (e). Fluorescence intensity tracks of the whole droplet and half droplet bleach (f). (g) Fluorescence images of HSF1 droplets under HS (42 °C, 0.5 h) condition before and after treatment with 10% 1,6-hexanediol for 1 min. Scale bars, 10 μm. (h) Inducing of HSF1 phase separation by Opto-Droplet in living cells under NHS condition. Representative fluorescence images showing the distribution of HSF1 before and after blue light activation. Scale bar, 10 μm. (i) Western blot showing slower migration of HSF1–HaloTag in the successful knock-in cells. GAPDH was used as a loading control. (j,k) Recruitment of HSF1 to LacO array can mediate the formation of a low-complexity domain (LCD) hub in living cells. Top, schematic for a LacO array (~256 LacO repeats) integrated into the genome of NIH3T3 cells. Bottom, HSF1-LacI-mCh formed an LCD hub after HS (42 °C, 0.5 h) when transiently expressed. Alternatively, LacI-mCh and HSF1-LacI-mCh were transiently expressed under NHS condition as controls. Scale bars, 5 μm (j). The mean fluorescence intensity at LacO foci in cells with different expression levels for the LacI-mCh (NHS), HSF1-LacI-mCh (NHS) and HSF1-LacI-mCh (HS) were measured (k). n = 22 cells for LacI, 21 cells for HSF1-LacI-NHS, and 38 cells for HSF1-LacI-HS pooled from 3 independent experiments. The data was smoothed in R using lm() function for linear smooths, and error bands represent the standard error of the smoothing. (l) EMSA showing that 1,6-hexanediol does not disrupt HSF1–DNA interaction. Images are representative of three independent experiments (b, e, g-j, l).
