Pu h71

The wild-type and mutant full-length Htt expression vectors GFP-C2-Htt 480-17Q-HA and GFP-C2-Htt 480-68Q, express the green fluorescent protein and Huntingtin with 17 and 68 glutamines respectively, and were kind gift of Dr. Humbert S. (Institute Curie-UMR 146 du CNRS, Centre Universities Orsay, France). For convenience, when we mention in the text, 17Q or 68Q, we refer to the plasmids GFP-Htt-480-17Q, and GFP-Htt-480-68Q respectively. Differentiated SH-SY5Y cells were transiently transfected with 17Q or 68Q vectors using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. At 6 h post-transfection, cells were treated with 1 μM GA (Sigma, G3381) or 50 nM PU-H71 (TOCRIS bioscience, 3104) for 24 h. In addition, the analysis of the expression of the Huntingtin protein is carried out by fluorescence of the green protein.

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).

Antibodies used: GAPDH (HyTest Ltd., clone 6C5, cat. # 5G4), caspase-3 (Abcam, ab4051), Cdk6 (Abcam, ab3126), Ras (Stressgen, now Enzo Life Sciences; ADI-KAP-GP001), p-ERK (Santa Cruz, sc-7383), Hsp70 (Abcam, ab181606), p27Kip1 (Cell Signaling, #2552), Nr2f1 (ABGENT, #AP14218a), tubulin (Merck, CP06), Cdk4 (NeoMarkers, MS-616-P1), Raf-1 (Santa Cruz Biotechnology, sc-133), Akt (Cell Signaling, #9272). Transfection reagents: jetPRIME (Polyplus, #114–15), polyethylenimine (PEI) (Polysciences, # 621405). Hsp90 inhibitors: geldanamycin (LC Laboratories, G-4500), PU-H71 (Tocris, #3104), pochoxime A (kindly provided by Nicolas Wissinger, University of Geneva). Other inhibitors: AZ628 (Tocris, #4836), PD98059 (Tocris, #1213), cycloheximide (Sigma, C7698), rapamycin (LC Laboratories, R-5000), aphidicolin (ACROS, BP615-1), hydroxyurea (fluorochem, #043351). All compounds were dissolved in DMSO. Growth factors: insulin (Sigma, I6634), recombinant human epidermal growth factor (EGF) (Lonza, CC-4017). Metabolic assay reagents: Oligomycin (SIGMA, 7535), carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) (SIGMA, C2920) and rotenone (SIGMA, R8875) with antimycin (SIGMA, A8674).