Extended Data Fig. 10: Negative regulation of HSF1 phase separation during HS is HSP70-specific.
(a) Representative images of HSP70 FL, HSP70-N, and HSP70-C phase separation. Recombinant HSP70 WT (10 μM), HSP70 N (10 μM), and HSP70 C (10 μM) were added to phase separation buffer with 200 mM NaCl in the presence (top) or absence (bottom) of 10% Dextran-2000. Scale bars, 2 μm. Images are representative of 3 independent experiments. (b) HSP70 FL (8 μM) was added to pre-formed HSF1-mCh (4 μM) droplets, the HSF1 droplets were dissolved rapidly in vitro. Scale bars, 2 μm. Images are representative of 3 independent experiments. (c) Representative images of HSF1-mCh (4 μM) with indicated concentration of HSP70 FL without ATP (left) and with 5 mM ATP (right). Scale bars, 2 μm. Images are representative of 3 independent experiments. (d) Analysis of HSF1 droplet size with different HSP70 concentrations (4 μM or 8 μM) with or without ATP. n = 2397/HSF1 + 4 μM HSP70-WO ATP (droplets/group), 0/HSF1 + 8 μM HSP70-WO ATP, 820/HSF1 + 4 μM HSP70-With ATP, 768/HSF1 + 8 μM HSP70-With ATP pooled across 3 independent experiments. Boxplots: 25th to 75th percentiles, median, 1.5×interquartile as whiskers. (e) Representative images of HSF1-mCh (4 μM) with the 8 μM HSP40 and HSP90 in vitro. Scale bars, 2 μm. Images are representative of 3 independent experiments.
