Fig 2: The HSF1 inhibitor KRIBB-11 and the apoptosis inducer ABT-263 reduce the viability of iCCA organoids. A, B Effect of ABT-263, KRIBB-11, and their combination on the viability of iCCA organoids, treated for 10 days in the specific medium of the hepatic organoid. Experiments were repeated three times in triplicate. ***P < 0.001, as calculated with a One-way ANOVA test

Fig 3: The HSF1 inhibitor KRIBB11 decreases the mitochondrial bioenergetics in cholangiocarcinoma cell lines. A Cell Mito Stress test profiles of human HuCCT1 and KKU-M156 cell lines treated with 10 µM KRIBB11 and matching DMSO concentration for 24 h. Seahorse XF Cell Mito Stress Tests (Agilent) were performed employing serial injections of oligomycin, FCCP, and Rot/AA; Hoechst 33342 was injected last for nuclei staining required for normalization. OCR was measured in real-time using the Seahorse XF HS mini analyzer. B Changes in basal respiration, maximal respiration and ATP production. Data were background corrected and normalized to the mean fluorescent intensity per well; a factor 105 was applied. Graphs depict the mean ± SEM of two independent experiments each performed in technical triplicates (multiple Mann–Whitney tests; *p < 0.05; **p < 0.01). Abbreviations: OCR, oxidative consumption rate (in pmol/min); FCCP, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; Rot/AA, rotenone/antimycin A

Fig 4: Impedance in response to Hsp90 inhibitors.A: Cells were treated 10 hours after seeding with different Hsp90 inhibitors. B: The effect of Hsp90 inhibition and protein synthesis inhibition by cycloheximide (CHX) 10 μg/ml. C: H1299 cells with HSF1 and HSF2 gene knockout. The impedance measurements were performed in four biological replicates, and the standard deviation of the measurements is expressed in the graph by the lighter color filling the area around the curve. D: Experimental setup showing correlation between the presence of denatured proteins after Hsp90 inhibition, impedance, and activation of HSF1.

Fig 5: Effect of proteosynthesis inhibition on HSF1 activation.A: H1299 cells expressing mCherry-HSF1 were exposed to 42°C heat shock, the proteasome inhibitor bortezomib (300 nM) and the Hsp90 inhibitor AUY-922 (300 nM) for 1 hour. HR CNE native electrophoresis shows inhibition of trimerization upon simultaneous inhibition of proteosynthesis by cycloheximide (CHX) 10 μg/ml, anisomycin 40 μM and rocaglamide 100 nM. B: HSF1 activation in living cells was determined by the detection of nuclear stress bodies. Nuclear distribution of mCherry-HSF1 was detected after 1 hour of treatment with AUY-922 (300 nM), bortezomib (300 nM), and heat shock at 42°C, with or without simultaneous application of cycloheximide (CHX). C: The effect of proteosynthesis on HSF1 activation was tested by fractionation of H1299 cells containing endogenous HSF1. Cells were exposed to 42°C, bortezomib (300 nM), or AUY-922 (300 nM) for 1 hour, with protein synthesis inhibitors cycloheximide (CHX) 10 μg/ml, 40 μM anisomycin (Anis), 10 nM rapamycin (Rap.), or 100 nM rocaglamide (Roc.). Cell lysates were fractionated into nuclear (N) and cytoplasmic (C) fractions. Activated HSF1 bound to DNA is detected in the nuclear fraction, while inactive monomers are present in the cytoplasmic fraction. The level of HSF1 in each fraction was determined by SDS PAGE and detected by blotting using anti-HSF1 mouse mAb c-5 (Santa Cruz Biotechnology Inc.). The efficiency of fractionation was monitored using PARP as a nuclear control and tubulin as a cytoplasmic control. D: Cell fractionation was also used to test the activation of mCherry-HSF1. The graph showing the relative amounts of mCherry-HSF1 in the nuclear (red bar) and cytoplasmic fractions (grey bar) was obtained by measurement of mCherry fluorescence. E: Effect of mTOR inhibitors on HSF1 activation at increasing concentrations of the Hsp90 inhibitor AUY-922. H1299 cells with endogenous HSF1 were pretreated with the mTOR inhibitors BEZ-235 and rapamycin (100 nM) for 8 hours. The effect of AUY-922 was monitored 2 hours after adding it at concentrations of 0–200 nM. HSF1 trimerization was detected by protein crosslinking by adding 2 mM DSBU (disuccinimidyl dibutyric urea) to the lysate for 30 minutes. HSF1 was resolved by SDS-PAGE and detected with mouse monoclonal antibody c-5. F: HSF1-mCherry trimers in identically treated cells were detected by HR CNE. See S1 Raw images for raw data.