Measuring Calcium Dynamics and Organelle Visualization

Cytoplasmic calcium concentrations were determined as previously described^{29 (link)}. Briefly, HeLa cells were labeled for 30 min with 5 μM Fura-2 and incubated at 37 °C/5% CO₂ in calcium-free Locke’s solution containing 154 mM NaCl, 5.6 mM KCl, 3.2 mM MgCl₂, 5 mM HEPES, 10 mM glucose, and 0.2 mM EGTA (pH 7.4). After drug treatment, changes in fluorescence were monitored every 5 min using an IX81 ZDC microscope (Olympus) at an emission wavelength of 510 nm, with dual excitation at 340 and 380 nm. Images of 340/380 fluorescence ratios were generated and analyzed using the Xcellence software package (Olympus). To detect HSF1 localization, HeLa cells were transfected with an HSF1–GFP plasmid^{30 (link)} obtained from Addgene (Addgene plasmid #32538) using the Lipofectamine transfection reagent (Invitrogen). After transfection for 24 h, cells were treated with the indicated drugs and then analyzed using an IX81 ZDC (Olympus). To visualize mitochondrial structure, HeLa cells were stained for 20 min with 200 nM MitoTracker dye, treated with the drugs, and then analyzed under an LSM 780 confocal microscope (Zeiss).