Radicicol

Ganetespib was obtained by Synta Pharmaceuticals (Lexington, MA, USA). ABT737 and ABT199 were kindly donated by Dr Vogler (University of Leicester, Leicester, UK). PU-H71 and Radicicol were purchased from Tocris Bioscience (Bristol, UK). 17-AAG was purchased from Sigma (St. Louis, MO, USA). The antibodies against PARP, BID, BIK, PUMA, CASPASE 8, BCL-2, BCL-Xl, BCL-w and GFP were obtained from Cell Signaling (Danvers, MA, USA), BAX, BAK and MCL1 antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA) and β-tubulin was obtained from Abcam (Cambridge, UK). Cytochrome-c antibody was purchased from BD PharMingen (Oxford, UK). Secondary antibodies were goat anti-rabbit HRP (DAKO, Glostrup, Denmark) and donkey anti-mouse HRP (GE Healthcare, Amersham, UK).

Geldanamycin (GA, Tocris) and radicicol (RA, Tocris) were stored in the dark at −20°C as stock solutions at 1.78 mM in dimethylsulfoxide (DMSO, Sangon) and ethanol, respectively. Cells were treated with either GA or RA at the concentrations as described in the text for different times. MG132 (Millipore) was stored at −20°C as a stock solution at 5 mM in DMSO. Cells were treated with MG132 (5 μM) for 2 h. For cycloheximide (CHX, Sigma-Aldrich) chase analysis, 100 μg/ml CHX was used for the indicated times as described in the text.

The chemical structures of the drugs used are depicted (Figure 1). Morphine (Sigma, 0.3 mg/kg IV bolus) was dissolved in saline. This dose was determined from prior studies using our rodent model, which included determining the dose-dependent effects of Morphine on myocardial infarct size [13 (link)]. The GSK3β inhibitor, SB216763 (Tocris, 0.6 mg/kg), was dissolved in DMSO (1 mg/mL, given in < 0.2 mL volume). The dose was selected based upon prior studies [6 (link), 14 (link)]. Two HSP90 inhibitors used were radicicol (Tocris, 0.3 mg/kg) and deoxyspergualin (DSG, 0.6 mg/kg) both dissolved in DMSO (1 mg/mL, given in < 0.2 mL volume). radicicol inhibits the ATP binding site at the N-terminus of HSP90 [5 (link)]. DSG interferes with the EEVD C-terminus sequence motif of HSP90, limiting protein interactions with the C-terminus [15 (link), 16 (link)]. The volume of DMSO delivered does not affect myocardial function as determined from our prior studies using this model [6 (link), 17 (link)].