17-AAG induces a heat shock response in OLN-A53T cells and does not impair proteasomal activity.

<p>(A) Determination of cytotoxic potential. Cells were exposed to different concentrations of 17-AAG as indicated. After 24 h, neutral red and MTT assays were carried out. Values represent the mean ± SEM of 16 microwells each of two independent experiments (n = 32). (B) Immunoblot analysis of heat shock protein induction. Cells were treated with 17-AAG (1–200 nM, 24 h), or subjected to heat shock (HS: 44°C, 30 min, 24 h recovery) or to MG-132 (MG: 1 µM, 24 h). Cell lysates were prepared and immunoblot analysis was carried out with antibodies against the individual proteins as indicated on the right. Co, untreated control. (C) Proteasomal activity was determined in cell lysates treated with 17-AAG (lysate) and in cell lysates prepared from 17-AAG treated live cells (cells). Cytoplasmic lysates were incubated with the proteasomal inhibitor MG-132 (1 µM, 60 min) as a positive control, or 17-AAG (50 nM, 60 min). Cells were treated with MG-132 (1 µM, 24 h) and 17-AAG (50 nM, 24 h). The post-glutamyl-peptidase-hydrolase activity was determined using fluorogenic substrate Z-Leu-Leu-Glu-AMC (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008753#s2" target="_blank">Materials and Methods</a>). The cleavage of the substrate is inhibited by MG-132 but not by 17-AAG. Data are expressed as percent of the untreated control and show the mean ± SEM from 3 independent experiments. Statistical evaluation was carried out by ANOVA/Fisher's LSD: ***p≤0.01 for MG-132 versus control.