Gal screen reagent

Strain CaLC922, containing the HSP70 promoter driving expression of the lacZ gene from Streptococcus thermophilus integrated at the HSP70 native locus, was grown overnight in YPD medium with 80 μg/ml uridine at 30°C with shaking, diluted to an OD₆₀₀ of 0.05, and grown with shaking for 5 h at 30°C with or without 10 μM geldanamycin, 40 μM MG132, or 0.2% DMSO as a vehicle control. To induce heat shock, overnight cultures were diluted to an OD₆₀₀ of 0.1, grown for 4 h at 30°C before addition of an equal volume of fresh YPD medium at 54°C, and immediately transferred to 42°C for incubation for 60 min with shaking. The induction of galactosidase activity was measured using a luminescent Gal-Screen reagent (catalog no. T1030; Applied Biosystems) at a reagent/cell ratio of 1:1, as per the manufacturer’s recommendations, and normalized to cell growth measured by the OD₆₀₀. Two independent experiments with three replicates were performed. Statistical significance was determined by averaging technical triplicates, and an unpaired t test was performed using GraphPad Prism, version 8.

Concentration-dependent activation of the heat-shock response in human cells by Hsp90 inhibitors was measured in 96-well format as previously published using 293T cells stably transduced with a reporter construct encoding GFP-luciferase fusion protein under transcriptional control of heat-shock elements from the HSP70B gene^{74 (link)}. Activation of the response in C. albicans was also measured in 96-well format using a reporter strain (CaLC922) engineered with HSP70 promoter elements driving expression of the LacZ gene from Streptococcus thermophilus integrated at the HSP70 locus with the URA3 marker for selection. After culture for 2 h in the presence of serial dilutions of Hsp90 inhibitor, the induction of galactosidase activity was measured using luminescent Gal-Screen reagent per manufacturer’s recommendations (Applied Biosystems; Bedford, MA Cat#T1029).