Bortezomib

U373, T98G, and U87 (human glioblastoma cell lines with mutant and wild type p53) and Panc1 (human pancreatic cancer cell line with mutant p53) were grown in RPMI 1640 (Thermo Fisher Scientific), 10% Fetal Bovine Serum (FBS) (Corning), L- glutamine, streptomycin (100 μg/ml) (Corning), and penicillin (100 U/ml) (Corning) in 5% CO₂ at 37°C. Cells were always detached using Trypsin-EDTA solution (Biological Industries, Cromwell, CT, USA).
U373, T98G, U87, and Panc1 cells were treated with AG490 (100 μM) (Millipore) for 48 h. U373 cells were treated with lovastatin (50 μM) (Sigma Aldrich) for 24 h. U373 cells were pre-treated with bortezomib (5 nM) (Santa Cruz Biotechnology) for 30 min and then treated with AG490 (100 μM) (Millipore) for 48 h. U373 and Panc1 cells were treated with geldanamycin (100 nM) (Sigma Aldrich) for 24 h.

Benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (Z-VAD-FMK) was purchased from Medical and Biological Laboratories (Nagoya, Japan). 1G244 was purchased from AK Scientific (Union city, CA). Alogliptin was purchased from ChemScene (Monmouth Junction, NJ). Linagliptin was purchased from BioVision (Milpitas, CA). Sitagliptin and Bortezomib were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Vildagliptin was purchased from LKT Laboratories (St. Paul, MN). Saxagliptin was purchased from Adooq Bioscience (Irvine, CA).

Yeasts were grown overnight in EMM2 medium at 30°C. Then, cells were pelleted (2,250 × g for 5 min) and resuspended in EMM2 at 0.05 OD600 (measured in an Infinite 200 PRO microplate plate reader, TECAN). Yeast cultures (200 μl) were inoculated in a 96-well plate (in triplicate), and cells were incubated for 24 h in constant agitation at 30°C in the plate reader, recording OD600 each hour. For thermal stress, cells were incubated at 42°C for 2 h, and then at 37°C for 22 h. For growth in the presence of the proteasome inhibitor Bortezomib (Santa Cruz Biotechnology), cells were prepared as previously described at a final OD600 of 0.05. Bortezomib was then added at a final concentration of 100 μM, or an equal quantity of DMSO (solvent) and cells were inoculated in 96-well plates in triplicate for each condition. Cells were grown for 24 h, and the OD600 was recorded. For the specific growth rate (μ index), the OD600 at the exponential phase of each strain was determined, and the values inserted into the following equation:
where k is the specific growth rate, N1 and N2 are the initial and terminal OD600 recordings, respectively, and t1 and t2 correspond to initial and terminal times, respectively (in hours). Thus, k is expressed in h⁻¹. The generation (or doubling) time was calculated according to the following equation:
where generation time is expressed in hours.

Activated T-lymphocytes were induced to undergo apoptosis by UV-B irradiation for 30 s (90 J/cm²) in the presence or absence of 1 nM bortezomib (Santa Cruz, Heidelberg, Germany) or 10 µM Y27632 (Merck, Darmstadt, Germany). After 20 h, the amount of released LEVs was quantified by flow cytometry (FSC/SSC analysis), using an EPICS XL™ flow cytometer (Coulter, Hialeah, Fl, USA). Cell viability was assessed by AxV/PI staining as described above.

Human colorectal carcinoma cell line HCT116 p53^+/+ and breast adenocarcinoma MDA-MB-231 cell line was from ATCC. HCT116 p53^−/− were kindly provided by Prof. B. Vogelstein (Johns Hopkins University School of Medicine) [27] (link).
Cells culture medium DMEM was from PAN Biotech (Aidenbach, Germany) and fetal bovine serum (FBS) was from Hyclone (Cramlington, UK). The proteasome inhibitor bortezomib was obtained from Santa Cruz Biotechnology (Santa-Cruz, USA). General caspase inhibitor Z-VAD-FMK and inhibitor to lysosomal cathepsins E-64 were from MP Biomedicals (Eschwege, Germany), Bafilomycin A1 was from Cayman Chemicals (Ann Arbor, USA). Neutralizing antibodies to TRAIL death and decoy receptors were from Enzo Life Sciences (Farmingdale, USA) and R&D systems (Minneapolis, USA), respectively. FITC-conjugated antibodies to DR4, DR5, DcR1 and DcR2 receptor were obtained from Abnova (Walnut, USA), isotype control antibody was from Immunotech (Marcelle, France). For western blot biotinylated goat antibodies to TRAIL death and decoy receptors were purchased from R&D Systems (Minneapolis, USA), HRP streptavidin and antibodies to actin were from Sigma-Aldrich (St. Lotus, USA).

Antibodies against USP7 (Cat. CST#4833), K48Ub (Cat. CST#4289), and K63Ub (Cat. CST#12930) were purchased from Cell Signaling Technologies, Inc. The antibodies against RNF6 (Cat. #20437-1-AP), USP9x (Cat. #55054-1-AP), PARP (Cat. #13371-1-AP), Caspase 3 (Cat. #19677-1-AP), GAPDH (Cat. #60004-1-Ig), α-tubulin (Cat. #11224-1-AP), and V5 (Cat. #14440-1-AP) were obtained from Proteintech. The monoclonal antibodies including anti-Flag (Cat. #M185-3L), anti-HA (Cat. #M180-3), and anti-Myc (Cat. #M192-3) were obtained from Medical and Biological Laboratories Co Ltd. The anti-Ub antibody (Cat. #SL-8017) was purchased from Santa Cruz Biotechnology, Inc. HRP-labeled goat anti-mouse (Cat. #A0216) and goat anti-rabbit IgG (H + L) antibodies (Cat. #A0208) were purchased from Beyotime Institute of Biotechnology. MG132 (Cat. #S2619), Bortezomib (Cat. #S1013), and P5091 (Cat. #S7132) were purchased from Santa Cruz Biotechnology and Selleck Chemicals Inc, respectively. CHX (Cat. #C7698) and chloroquine (CHQ, Cat. #C6628) were purchased from Sigma-Aldrich. LBH589 (LBH, Cat. #M1748) was purchased from AbMole Bioscience Inc. Nilotinib (Cat. #IN0560) was purchased from Solarbio Life Science. Recombinant ubiquitin (Cat. #U100-H), UBE1 (Cat. #E-304), UBE2D1(Cat. #E2-616), and ATP (Cat. #B-20) were all purchased from Boston Biochem Inc.