Q vd oph sml0063

Green-to-red FPX Caspase-3 reporter plasmids, GANES-DEVD-BNLS (50842), RANLS (50843) and single polypeptide FPX biosensor for caspase-3 (60883), and M50054 (ab145906) were purchased from Abcam (Cambridge, UK). MISSION® esiRNA’s targeting Apaf-1, Caspase-2 and Bid and control scrambled siRNAs and Q-VD-OPh (SML0063) were from Sigma-Aldrich (St. Louis, MO). Cycs Mouse siRNA Oligo Duplex (SR401266) was from OriGene (Rockville, MD). Plasmids, pcDNA3-Casp2-Flag (11811), pcDNA3-Casp2 C303A-Flag (11812), pCMV-BID (21131), pCMV-BID (D59E) (21133) and pCMV-tBID (21149) were purchased from Addgene. Details of primary and secondary antibodies, siRNAs and plasmids are provided in Supplementary Materials.

In order to assess the effect of PP1 and PP2A phosphatases, cells were treated for 2 h (unless otherwise stated) with 1 μM tautomycetin (TMC) (2305, Tocris) or 100 nM OA (ALX-350-003; Enzo Life Sciences), respectively. When phosphorylation of MCL-1 was studied, all samples were treated with 20 nM bortezomib (S1013; Selleckchem) and 10 μM Q-VD-OPh (SML0063; Sigma-Aldrich). Protein half-life determination was performed using 25 μg/ml cycloheximide (CHX) (HY-12320; MedChemExpress) for 0, 1, 2, and 4 h or for 0.5, 1, and 2 h. In case of CHX experiments with OA, cells were pretreated for 2 h with OA, and OA remained present during CHX treatment. In order to determine MCL-1 half-life in primary MM samples, CD138+ cells were purified from MM bone marrow aspirates by magnetic activated cell sorting using CD138 microbeads (Miltenyi Biotec), followed by CHX treatment as described above. CDK7 inhibitor THZ1 (MedChemExpress) was used at 200 nM for 8 h in order to inhibit MCL1 transcription. Ten micromolar of GSK-3β inhibitor CHIR99021 (Tocris Bioscience), 250 nM of MEK/ERK-1 inhibitor trametinib/GSK1120212 (Selleckchem), and 20 µM of JNK inhibitor SP600125 (Merck) were used to assess the role of kinases in MCL-1 degradation.