Z fa fmk

Novartis Pharma generously provided Smac mimetic LCL161. Vincristine was obtained from Sigma-Aldrich. Inhibitors targeting caspase-8 (Z-IETD-FMK), caspase-9 (Z-LEHD-FMK) and general caspase inhibitor (Z-VAD-FMK) were purchased from R&D. Negative Control for caspase inhibitors (Z-FA-FMK) was purchased from BD Pharmingen. Combined inhibition of canonical and non-canonical NF-κB activity was performed using PBS-1086 kindly provided by Profectus BioScience. Canonical NF-κB signaling was inhibited with BAY 11-7085 obtained from Merck Millipore. Necrostatin-1 (Nec-1) from StressMarq Biosciences Inc. was used to inhibit necroptosis and RIP1 kinase activity. Working solutions were prepared by dilution of drugs with medium or ddH₂O to designated concentrations.

Femur and tibia pairs were flushed to harvest cells from the bone marrow as previously described (Riley et al., 1991 (link)). Single cell suspensions were washed and counted for use in flow cytometry, cell sorting, or cell culture. When obtaining IL-7 expanded B-cell precursors, bone marrow cells were cultured at 1–2 × 10⁶ cells ml⁻¹ with 5 ng ml⁻¹ rmIL-7 in RPMI-1640 (Invitrogen, Carlsbad, CA, USA) supplemented with 10% FCS (low endotoxin; Sigma–Aldrich, St. Louis, MO, USA) plus 1% penicillin-streptomycin, 1% glutamine, and 5 × 10⁻⁵ m 2-mercaptoethanol.
In analysis of apoptotic mechanisms, a caspase 3 inhibitor, Z-DEVD-FMK (BD Biosciences), was used to block caspase 3 activity with Z-FA-FMK serving as negative control (BD Biosciences) (Ratliff et al., 2013 (link)). Cells were preincubated with inhibitor or control for 30 min. Cells were cultured for the indicated times and assessed for intracellular λ5 protein as described above. Added inhibitor or control was included to maintain original concentrations (50 mm) during culture.

TRAIL was commercially supplied (Enzo Life Sciences, Farmingdale, NY, USA) or produced from 293T cells as described.^{45 (link)} Briefly, the extracellular portion of human TRAIL (amino acids 95–281) was cloned into pBabe retroviral expression vectors to produce functionally active TRAIL,^{46 (link)} which was quantified by TRAIL ELISA according to the manufacturer’s instructions (Abcam, Cambridge, UK). Caspase inhibitors (BD Pharmingen, San Diego, CA, USA) were as follows: Z-IETD-FMK (caspase-8 inhibitor), Z-VAD-FMK (general caspase inhibitor), Z-FA-FMK (negative control), Z-LEHD-FMK (caspase-9 inhibitor) and Ac-DEVD-CHO (caspase-3/7 inhibitor). Bcl-2, Bcl-xL inhibitors ABT-263 and ABT-737 were purchased from Cayman Chemicals (Ann Arbor, MI, USA)

RPMI 1640 medium was purchased from GIBCO (Carlsbad, CA, USA). Fetal bovine serum (FBS) was purchased from JRH Biosciences (Lenexa, KS, USA), and heat-inactivated at 56 °C for 30 min prior to use. Glutamine, 2-mercaptoethanol (2-ME) and propidium iodide (PI) were obtained from Sigma (St. Louis, MO, USA). Fluorescein isothiocynate (FITC)-anti human annexin V and FITC-anti human active caspase-3, Z-DEVD-FMK (a caspase-3 inhibitor), Z-VAD-FMK (a general caspase inhibitor), Z-FA-FMK (a negative control for Z-DEVD-FMK and Z-VAD-FMK), and Cytofix/cytoperm solution were purchased from BD Pharmingen (San Diego, CA, USA). Carbaryl, maneb, thiram and ziram were obtained from Wako Pure Chemical Industries (Osaka, Japan) and prepared as stock solutions in DMSO.

Human NK cells were isolated from peripheral blood lymphocytes of unidentified donors (NIH blood bank) by negative selection using the EasySep™ human NK cell enrichment kit (STEMCELL Technologies). Purified NK cells were co-cultured with an equal number of Mitomycin C (Roche Diagnostics)-treated autologous PBL feeder cells in IMDM (Life Technologies) supplemented with 10% human AB serum (Sigma-Aldrich), 10% purified IL-2 (Hemagen Diagnostics), 200 U/ml recombinant human IL-2 for one week, and then expanded NK cells were cultured with IMDM supplemented with 10% human AB serum and rIL-2 (200 U/ml). All cell lines were from the American Type Culture Collection (Manassas, VA, USA). Recombinant human IL-2 was from the National Cancer Institute-Frederick Cancer Research and Development Center (Frederick, MD, USA). Actinomycin D (ActD), Camptothecin (CPT) and Etoposide (ETO) were from Sigma-Aldrich. Z-VAD-FMK and Z-FA-FMK were from BD Biosciences. The synthetic metalloproteinase inhibitor BB-94 (Batimastat) was from Santa Cruz Biotechnology.