Marizomib

The following drugs were used: camptothecin (CPT), actinomycin D, cycloheximide, marizomib, chloroquine, Bmh21, BO-2, (all Sigma-Aldrich), aphidicolin (Santa Cruz), talazoparib (Axon Medchem), quarfloxin (CX-3543, Adooq Bioscience) and CX-5461 (Selleckchem).

Peptide FRET substrates with ten glutamine residues were procured from GenScript (Piscataway, NJ, USA); the substrate with five glutamine residues was purchased from Peptide Protein Research Ltd. (Fareham, UK). The standard fluorescent substrates for proteasome assays (N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin, Suc-LLVY-AMC) and HIV protease substrate 1 (Arg-Glu(EDANS)-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln-Lys(Dabcyl)-Arg) were from Sigma-Aldrich (St. Louis, MO, USA) and Invitrogen (Carlsbad, CA, USA), respectively. Ac-RLR-AMC and Z-LLE-AMC were from UBPBio (Dallas, TX, USA). Bortezomib was from LC Laboratories (Woburn, MA, USA), MG-132 (Z-LLL-CHO), leupeptin (as hemisulfate salt), and marizomib was from Sigma-Aldrich, and Z-P-nLeu-D-CHO was from Enzo Life Science (Farmingdale, NY, USA). All other reagents were of the highest quality available or for molecular biology grades and were used without additional purification.

Primary human pulmonary artery smooth muscle cells (HPASMCs) were obtained from ScienCell Research Laboratories (ScienCell Research Laboratories, Carlsbad, CA, USA) and were cultured in a smooth muscle cell medium (cat. #1101, ScienCell Research Laboratories, Carlsbad, CA, USA) supplemented with 2% fetal bovine serum, 1% smooth muscle cell growth supplement, and 1% penicillin–streptomycin in a humidified incubator at 37 °C containing 5% CO₂. HPASMCs were passaged at 70% to 80% confluence by dissociation from plates with 0.25% trypsin–EDTA and passages 5 to 12 were used in the experiment. When performing hypoxia experiments, HPASMCs were incubated under 1% Oxygen. The concentration of oxygen was controlled by an oxygen controller (model ProOx 110, BioSpherix Ltd., Redfield, NY, USA), which senses oxygen inside the chamber and infuses either nitrogen to reduce the concentration or oxygen to raise the concentration. Cobalt chloride (CoCl₂), bortezomib, and marizomib were purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA).

Ac-nLPnLD-amc, Boc-LSTR-amc and Suc-LLVY-amc peptides were purchased from Bachem AG (Bubendorf, CHE). Marizomib, chloroquine and verteporfin were from Sigma (St. Louis, MO), bortezomib was from Tebu bio (Le Perray en Yvelines, France), bafilomycin A1 and everolimus were from Invivogen (San Diego, CA, USA).