on assay (MTT assay) was carried out on NSC34 cells in 24-well multiwell at 80,000 cell/mL density, treated with different 17-AAG doses. To this purpose, MTT (Sigma- Aldrich) solution was prepared at 1.5 mg/ml in RPMI without phenol red and was ?ltered through a 0.2-  m ?lter. Then, the culture medium was removed from the plate and 300  l of MTT solution was added into each well. Cells were incubated for 30 min at 37 C with 5% CO 2 , 95% air and complete humidity. After 30 min, 500  l of 2-propanol was added into Paclitaxel each well and the solution was resuspended. The optical density (OD) of the wells was determined using a plate reader at a wavelength of 550 nm. mRNA expression analysis The cells were transfected as described above. 48 h from trans- fection, cells were harvested in 4 M guanidium isothiocyanate (containing 25 mM sodium citrate pH 7.5, 0.5% sarcosyl and 0.1% 2-mercaptoethanol) total RNA isolated by phenol-chloroform extraction according to Chomczynski and Sacchi (1987) .

RNA quan- ti ?cation was carried out by absorption at 260 nm. Total RNA (1  g) was treated with DNAse and reverse transcribed into cDNA using the High-Capacity cDNA Archive Kit (Applied Biosystems) according to the manufacturer’s protocol, with the following reaction. Primers for real-time RT-PCR of the LC3 B mRNA were designed in accordance with recommendations accompanying the ABI Prism 7700 Sequence Paclitaxel 33069-62-4 Detection System (Applied Biosystem, CA, USA) on C-G base content and 5′ content and using the program Primer Express 1.5. The primers were synthesized by MWGBiotech (Ebersberg, Germany) with the following sequences: mMAP-LC3B

5′-CAG TGA GCT TCC CGT TCA- 3′ (reverse). The evaluated ef ?ciency of each set of primers was close to 100% for both target and reference gene. Real-time PCR was performed using the ABI Prism 7000 sequence detection system (PE Applied Biosystems) in a 25-  l total volume, using the iTaq SYBR Green Supermix (BioRad Laboratories), and with 500 nmol primers. PCR cycling conditions were as follows: 94 C for 10 min, 35 buy Paclitaxel cycles at 94 C for 15 s, and 60 C for 1 min. Melting curve analysis was always performed at the end of each PCR assay to control for speci ?city. Data was expressed as Ct values and used for the relative quanti ?- cation of targets with the  Ct calculation. To exclude potential bias due to averaging data transformed through the equation 2 ? Ct to give N-fold changes in gene expression, all statistics were performed with  Ct values. Each experiment was carried out with four independent samples. LC3 values were then normalized with those of GAPDH. Proteasome activity The cells were transfected as described above. Proteasome assays were performed as described by Allen and co-workers ( Allen et al., 2003 ).

Cells were washed with ice-cold PBS and then harvested and centrifuged at 1200 rpm for 5 min, at 4 C. The pellets were resuspended in 0.3 mL of proteasome extract buffer (20 mM Tris/HCl, pH 7.4, containing 0.1 mM EDTA, 1 mM 2-mercaptoethanol, 5 mM ATP, 20% v/v glycerol, and 0.04% v/v Nonidet P-40). The resuspended cells were pork belly homogenized by repeated passages through a 21-gauge needle. The homogenates were centrifuged at 13,000 g for 15 min at 4 C. Total proteins were determined with BCA assay (Pierce). Proteasome assay reaction mixtures consisted of 50 mM HEPES/KOH, pH 8.0, containing 5 mM EGTA, 100  g of cells extract protein per ml of assay reaction. The reactions were initiated by adding the appropriate proteasome substrate and incubating the reactions at 37 C for 45 min. Suc-LLVY-AMC was used at 50  M. Z-ARR-AMC and Z-LLE-AMC were used at 100  M. Hydrolysis of the peptides was measured at 340 nm excitation and 460 nm emission for AMC using a spectro ?uorimeter (VICTOR3 1420 multilabel plate reader, Perkin Elmer). Statistical analysis Statistical analysis has been performed using one-taile