Figure 1.

Celastrol activates yeast Hsf1 and induces thermotolerance. (A) Wild-type (BY4741) cells containing an HSE-lacZ reporter were treated with concentrations of celastrol ranging from 0 to 40 μM for 1 h, and β-galactosidase activity was determined as described in Materials and Methods. (B) The same cells as in A were grown at control temperature (30°C, −), heat shocked at 37°C for 1 h (HS), or exposed to DMSO or celastrol (cel, 20 μM), and β-galactosidase activities were determined. (C) W303 HSF1-TAP cells exposed to normal growth temperatures (30°C), heat shock (39°C), DMSO, or 10 μM celastrol were harvested at the indicated time points. Protein extracts were analyzed by SDS-PAGE and immunoblot analysis and Hsf1-TAP detected using antibodies directed against the protein A epitope. Extract prepared from W303 cells grown under normal growth conditions was used as a negative control (−). (D) Wild-type (BY4741) cells were exposed to 30°C, 39°C, DMSO, or 20 μM celastrol for 1 h, and protein extracts were analyzed by SDS-PAGE and immunoblot. Ssa3/4 levels were detected using polyclonal antibodies that detect both heat shock-inducible isoforms of the yeast Hsp70. PGK levels were determined as a loading control. (E) Wild-type (BY4741) cells were treated for 1 h with either no reagent, DMSO, or 10 μM celastrol for 1 h before heat shock at 47°C for the indicated times as described in Materials and Methods. Cells were spotted in equal volume on SC medium, and viability was assayed after growth at 30°C for 2 d.