Z vad fmk z vad

HCT116 p21+/+, HCT116 p21−/−, U2OS and MDA-MB-231 cells were cultured as instructed. To compensate the faster proliferation HCT116 p21+/+ cells were seeded 10% less than HCT116 p21−/− (except: proliferation assays). BI 2536 and BI 6727 were purchased from Selleck Chemicals LLC (Houston, USA). The pan-caspase inhibitor Z-VAD-FMK (Z-VAD) was obtained from Enzo Life Science GmbH (Lörrach), DMSO from Sigma-Aldrich (Taufkirchen), PD98059 from Merck Millipore (Darmstadt) and wortmannin from Cell Signaling (Beverly, USA). siRNA (20 nM) transient transfections were performed as previously described [7 ]. Regarding Plk1 depletion, two different siRNAs (each 10 nM) were mixed to avoid off-target effects. siRNAs targeting p21 (sense: ACACCUCCUCAUGUACAUAUU and antisense: AAUAUGUACAUGAGGAGGUGU; designated as sip21 #2) or Plk1 (sense 1: GAUCACCCUCCUUAAAUAU and antisense 1: AUAUUUAAGGAGGGUGAUC, sense 2: GAAGAAUGAAUACAGUAUU and antisense 2: AAUACUGUAUUCAUUCUUC) were manufactured by Sigma-Aldrich. A different siRNA against p21 (referred to as sip21 #1), containing a pool of siRNAs, was from Santa Cruz (Heidelberg). Control siRNA was obtained from Qiagen (Hilden). For irradiation, cells were exposed to a single dose of 2.0 Gy at room temperature by a linear accelerator (SL 75/5, Elekta, Crawley, UK) with 6 MEV photons/100 cm focus-surface distance and a dose rate of 4.0 Gy/min.

The following reagents were obtained as indicated: zVAD-FMK (ZVAD) (ENZO Life Sciences), recombinant human TNF-α and IL-1β (BioLegend), poly(I:C) (HMW) (Invivogen), doxorubicin (Calbiochem), DAPI (Dojindo), blasticidin (Wako), pepstatin A, chloroquine, and cycloheximide (Sigma), E64d (Tokyo Chemical Industry), monoubiquitin, eight kinds of diubiquitins, linear (M1)-, K11-, K48-, and K63-tetraubiquitins (Boston Biochem), control siRNA (sc-37007) and NDP52-siRNA (sc-93738) (Santa Cruz Biotechnology), and BV6 (Genentech). HOIPIN-1 (2-[(1E)-3-(2-methoxyphenyl)-3-oxoprop-1-en-1-yl] benzoic acid sodium salt) and HOIPIN-8 (2-{(E)-3-[2,6-difluoro-4-(1H-pyrazol-4-yl)-phenyl]-3-oxo-propenyl}-4-(1-methyl-1H-pyraol-4-yl)-benzoic acid sodium salt) were prepared as described (11 (link), 12 (link)).

TAK1,^{56 (link)} F4/80 (BM8, eBioscience, San Diego, CA, USA), CD16/32 (93, BioLegend, San Diego, CA, USA), CD4 (RM4-5, BioLegend), CD8a (53-6.7, BioLegend), CD11b (M1/70, BioLegend), Ly6C (HK1.4, BioLegend), Ly6G (1A8, BioLegend), cathepsin B (FL-339, Santa Cruz, Dallas, TX, USA), Lamp1 (Anti-Human CD107a, eBioscience or H4A3, Santa Cruz), and β-actin (AC-15, Sigma, St. Louis, MO, USA) antibodies were used. Human recombinant TNFα (Peprotech, Rocky Hill, NJ, USA), Z-VAD-FMK (Z-VAD) (Enzo Life Sciences, Farmingdale, NY, USA) and acridine orange (ThermoFisher Scientific, Waltham, MA, USA) were used. The TAK1 kinase inhibitor, 5Z-7-oxozeaenol (5Z) was described previously.^{38 (link)}