Figure 6

Celastrol upregulates the expression of heat-shock response genes, which delay cell death in human glioblastoma cells. (a) U251N cells were treated with serum-containing or serum-deprived media in the presence and absence of 1 μg/ml CHX, an inhibitor of protein translation, for 1 h, after which cells were treated with celastrol for 3–24 h. CQ (50 μM) for 24 h controls for protein accumulation following inhibition of autophagy. Total cell lysates were collected and immunoblotted for Hsp72. Actin is used as a control for protein loading. (b) U251N cells were treated with serum-containing and serum-deprived media in the presence and absence of 500 nM 17-AAG (Hsp90 inhibitor) or 10 μM MG-132 (proteasome inhibitor) for 24 h and 3 μM celastrol for 3–24 h. Total cell lysates were collected and immunoblotted for Hsp90. Actin is used as a control for protein loading. (c) U251N cells were treated with serum-deprived media with or without 500 nM 17-AAG for 1 h, after which celastrol was added at various concentrations (0.3–10 μM) for another 24 h. Cell viability was assessed using the MTT assay. 17AAG significantly shifted the dose–response to celastrol treatment relative to treatment with celastrol alone (LD50: 1.03±0.12 μM without 17-AAG versus 0.69±0.11 μM with 17-AAG; P=1.23 × 10⁻⁶). Average values and S.Ds. are reported for triplicate measurements (N=3). Results are representative of at least three independent experiments