Figure 5

Celastrol promotes proteotoxic stress in a panel of human glioblastoma cell lines and GSCs. (a) U343 and (b) U87 cells were treated with serum-containing and serum-deprived media in the presence and absence of 50 μM CQ (inhibitor of lysosomal acidification), 500 nM 17-AAG (Hsp90 inhibitor), and 3 μM celastrol for 24 h. Total cell lysates were collected and immunoblotted for proteasomal substrates (ubiquitin) and autophagy substrates (p62). Actin is used as a control for protein loading. (c) U343 and (d) U87 cells were treated with serum-deprived media with or without 1 μg/ml CHX, an inhibitor of protein translation, 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. A significant difference in the response to celastrol was noted with CHX-treated U343 cells relative to treatment with celastrol alone (LD50: 5.30±0.28 μM without CHX versus 7.32±0.1 μM with CHX; P=8.36 × 10⁻⁶) (see Statistical analysis section in Materials and Methods). Average values and S.Ds. are reported for triplicate measurements (N=3). (e) GSCs were treated with serum-deprived media with or without 1 μg/ml CHX 1 h, after which celastrol (3 μM) was added for another 24 h. Cell viability was assessed using the MTT assay and reported as the percentage of control (untreated cells for celastrol−CHX and CHX-treated cells for celastrol+CHX). A significant difference in the response to celastrol was noted with CHX-treated GSCs as noted in the annotations. At right, the response to 1 μg/ml CHX alone is observed to reduce cell viability and proliferation by∼30–40%. Average values and S.Ds. are reported for triplicate measurements (N=3)