Caspase 8 inhibitor z ietd fmk

Human bone marrow‐derived mesenchymal stromal cells from eight different healthy male donors (MSC, Lonza, Walkersville, Maryland, donors lot #; 1F3422, 318006, 1F4287, 307219, 374385, 351482, 422610, 8F3520) were cultured in mesenchymal stem cell growth media (MSCGM) (Lonza) at 37°C in a humidified atmosphere containing 5% CO₂ according to the manufacturer's instructions. The cells were passaged for a maximum of seven times. For experiments, 70 000 hBMSCs were seeded per cm². At 80% confluency the cells were treated as indicated in the figure legends with TLR2/TLR1 agonist Pam₃Cys‐Ser‐Lys₄ (Pam₃CSK₄) (1 μg/ml) (EMC microcollections GmbH, Tuebingen, Germany), the TLR3 agonist polyinosinic–polycytidylic acid (poly(I:C)) (5 μg/ml) (Amersham Life Science, Little Chalfont, United Kingdom), TLR4 agonist lipopolysaccharide (LPS) obtained from E. coli O111:B4 (LPS B4) (1 μg/ml) (InvivoGen, SanDiego, California) and ultrapure preparations of LPS from E. coli K12 (LPS K12) (1 μg/ml) (InvivoGen). LPS B4 was used in generating the data in Supplementary Table S1, LPS K12 was used in all other experiements. Caspase‐8 Inhibitor z‐IETD‐fmk (R&D Systems, Minneapolis, Minnesota) was added to the cells 2 h before cells were treated with TLR agonists.

CellTiter 96 AQ_ueous One Solution Cell Proliferation Assay Reagent [MTS, 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] was purchased from Promega (Madison, WI, USA). Propidium iodide (PI) and 4′,6-diamidino-2-phenylindole (DAPI) stain were purchased from Sigma-Aldrich (St. Louis, MO, USA). NE-PER Nuclear and Cytoplasmic Extraction Reagents were purchased from Pierce (Rockford, IL, USA). Antibodies specific to PARP, caspase-3, caspase-8, p53, Bcl-2, Bcl-xL, Bax, Bid, pRb, p-pRb, and cytochrome C were purchased from Cell Signaling Technology (Beverly, MA, USA). The anti-rabbit IgG horseradish peroxidase (HRP)-conjugated secondary antibody and anti-mouse IgG HRP-conjugated secondary antibody were purchased from Millipore (Billerica, MA, USA). Antibodies specific to p27, p21, and glyceraldehyde 3-phospahte dehydrogenase (GAPDH) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl benzimidazolycarbocyanine chloride) was purchased from Enzo (Farmingdale, NY, USA). General-caspase inhibitor Z-VAD-fmk and caspase-8 inhibitor Z-IETD-fmk were purchased from R&D systems (Minneapolis, MN, USA). The FITC-Annexin V Apoptosis Detection Kit I was purchased from BD Biosciences (San Jose, CA, USA).

Halofuginonehy drobromide, 5-fluorouracil, Earle's Balanced Salt Solution (EBSS), chloroquine (CQ), propidium iodide (PI) and Pierce (R) BCA Protein Assay Kit were obtained from Sigma-Aldrich (Munich, Germany). Artemisinin and In situ BrdU-red DNA fragmentation (TUNEL) assay kit were purchased from Abcam (Cambridge, UK). Antibodies against cleaved capase-8, cleaved capase-9, cleaved caspase-3, cleaved PARP, SQSTM1/p62, LC3-II and β-Actin were purchased form Cell Signaling Technology (Danvers, MA, USA). HRP-goat anti-rabbit secondary antibody was purchased from Invitrogen (Carlsbad, CA, USA). Goat antimouse IgG-HRP secondary antibody and caspase-3 inhibitor (z-DEVD-fmk) were purchased from San Cruz Biotechnology (Santa Cruz, CA, USA). Caspase-8 inhibitor (z-IETD-fmk) and caspase-9 inhibitor (z-LEHD-fmk) were purchased from R&D Systems (Wiesbaden-Nordenstedt, Germany). FITC Annexin V Apoptosis Detection Kit I was obtained from BD Bioscience (San Jose, CA, USA).