Qvd oph

We used different pharmacological inhibitors to determine the cell death mechanism of I-CRP in leukemic cells. QVD-OPh (QVD, 10 μM) as a general caspase inhibitor; Z-DEVD (1 μM) as caspase-3 inhibitor; Z-IETD (1 μM) as caspase-8 inhibitor; and Z-LEHD (1 μM) as caspase-9 inhibitor were acquired from BioVision (CA, USA). N-acetyl-cysteine (NAC, 5 mM) was used as an ROS inhibitor. The inhibitors were added 30 minutes before CC₅₀ I-CRP treatment. All stock solutions were wrapped in foil and stored at −20°C.

MTT reduction assay was performed as previously described (Damgaard et al, 2013) with cells treated with recombinant human TNF (100 ng/ml; Life Technologies), ultrapure LPS from E. coli K12 (100 ng/ml; Invivogen), poly(I:C) (1 μg/ml; Invivogen), staurosporine (1 μM; Sigma), cycloheximide (CHX; 50 μg/ml; Santa Cruz Biotechnology), or a combination of recombinant human TNF and CHX with or without the caspase inhibitor Q‐VD‐OPh (10 μM; BioVision, Milpitas, CA) or the RIPK1 kinase inhibitor necrostatin‐1 (Nec‐1) (10 μM; Cayman Chemical, Ann Arbor, MI) as indicated. For further details, see Appendix Supplementary Methods.

Two BRAF-mutated cell lines, A-375 [73 (link)] and Mel-HO [74 (link)], as well as two BRAF-WT melanoma cell lines, MEWO [75 (link)] and SK-MEL-23 [76 (link)], were cultured at 37 °C, 5% CO₂ in DMEM (4.5 g/L glucose; Invitrogen, Karlsruhe, Germany), supplemented with 10% fetal calf serum and antibiotics. For control, three different cultures of normal human fibroblasts were used (ATCC, Manassas, VA, USA), which were cultured in RPMI 1640 growth medium + 10% FCS.
Cells were treated with the selective BRAF (V600E) inhibitor vemurafenib/PLX4032 (Selleck Chemicals, Houston, TX, USA), the ERK1/2 inhibitor SCH772984 (Cay19166; Cayman Chemical, Hamburg, Germany), and the Mcl-1 inhibitor S63845 (HY-100741; MedChemExpress, Cologne, Germany), while control cells received the solvent DMSO. For caspase inhibition, the pan-caspase inhibitor QVD-Oph (Abcam, Cambridge, UK; 5 μM) was applied at 1 h before cells were treated with other agents. Most analyses were performed in 24-well plates, and 5 × 10⁴ cells were seeded per well.

SIM-A9 and 4T1 cultures were prepared by seeding 5x10⁴ cells/well in 24-well dishes (CORNING), then incubated with EPI or CTX alone or pre-treated for 30 min with 10 mM QVD-Oph (Abcam) (pan caspase inhibitor). Cells were then detached and stained using a General Caspase Activation Kit (TF2-VAD-FMK) (Abcam) following the manufacturer's instructions. Caspase activity was determined by flow cytometry and analyzed using FlowJo Software.