Extended Data Fig. 3: Time-lapse microscopy of tagged HSF1 in tissue culture cell lines.

a, Western blot on U2OS cell lines expressing fluorescent protein tagged HSF1. Lanes: 1) U2OS, 2) U2OS HSF1-mVenus, 3) U2OS HSF1-mEGFP, 4) U2OS HSF1-mTQ2, 5) U2OS HSF1Δp-mTQ2, 6)-7) U2OS HSF1-mVenus FKBP-cHSF1-mTQ2,6) no Shield-1, 7) 500nM Shield-1 for 24 hours. Top part of membrane, anti-HSF1 monoclonal antibody. Bottom part of membrane, anti-GAPDH antibody (see Unprocessed Blots Extended Data Fig. 3 for unprocessed blot image). b, Quantification of bands in lanes 2-4 from western blot image in a (see Methods for Quantification details). c, Left, kernel density estimate (KD) pdf of HSF1 antibody signal level in cells from 9 plasmacytoma/myeloma patient samples from Fig. 1e–k (total of n ~ 2*10⁶ cells). Right, KD pdf of HSF1 antibody level in U2OS (blue) and U2OS HSF1-mVenus cells (orange), n ~ 17,000 and 20,000 cells. d, Fold induction of HSPA1B, HSPA6, and HSP90AA1. RT–qPCR from bulk cell populations in U2OS, U2OS HSF1-mEGFP 3 hours post MG132 2.5uM (bars are biological replicates). e,f, Time-lapse microscopy of U2OS HSF1-mVenus cells treated with 42 °C temperature (scalebar = 10 µm) and quantification (n ~ 16,000-24,000 cells, median + 75th percentile, see Source Data Extended Data Fig. 3 for exact cell numbers and single cell data). f, Single-cell HSF1-FI quantification. g, Dynamics of the percentage of cells with foci after proteotoxic stressor addition (U2OS HSF1-mVenus cells, live cell time-lapse microscopy, 30 minutes intervals, n ~ 260 cells, see Source Data Extended Data Fig. 3), using three different thresholds to HSF1-FI to define the foci positivity, 0.025, 0.05 and 0.1. h, Fold induction of HSP70 genes (HSPA1B, HSPA6) and HSP90AA1 using RT–qPCR from bulk cell populations after gradient doses of MG132 and STA9090 (MG132: 5, 2.5, 1.25, 0.625 µM; STA9090: 50, 25, 12.5, 6.25 nM). Statistics and Source Data are provided in Source Data Extended Data Fig. 3.

Source data