Pd150606

ABT-263 was purchased from Selleck Chemicals (Houston, TX, USA). CTG was from Promega Life Sciences (Madison, WI, USA). AlamarBlue, Hoechst 33342, Fura-2-AM, and Alexa Fluor 488 Annexin V solution were from ThermoFisher (Pittsburgh, PA, USA). PD150606 was from Sigma-Aldrich. Z-VAD-FMK was purchased from Enzo Life Sciences (Farmingdale, NY, USA) and q-VD-OH was a generous gift of Shigemi Matsuyama at Case Western Reserve University, Cleveland, OH, USA.

A normal human liver cell line (QSG‐7701) and liver cancer cells (Hep3B, MHCC97H, HepG2 and HCCLM6) were purchased from American Type Culture Collection (ATCC). The other cells were a gift from Dr. Qu and Dr. Chen. A normal human liver cell line (QSG‐7701) was cultured in RPMI‐1640 medium (HyClone), and cancer cell lines were cultured in Dulbecco's Modified Eagle Medium (DMEM) (HyClone) containing 10% foetal bovine serum (Biological Industries). Moreover, these cells were cultured in an incubator at 37°C with 5% CO₂.
Necrostatin‐1, PD150606 and Z‐VAD‐FMK were obtained from Sigma Aldrich. The microtubule (MT)‐stabilizing agents epothilone B and paclitaxel and the MT‐destabilizing agents fosbretabulin and nocodazole were purchased from MedchemExpress Inc. Each of the reagents was dissolved in DMSO (Sigma Aldrich) and diluted to the appropriate concentrations.

BAPTA-AM and PD150606 were obtained from Sigma and were dissolved in DMSO before dilution in M9. We treated animals with drugs as previously described (Ghosh-Roy et al., 2010 (link); Xu and Chisholm, 2011 (link)). For Western blot, we incubated mixed-stage animals in the drug solutions (containing Escherichia coli OP50) for 24 hr before experiments; for quantification of RME defects, we grew L1 animals in the drug solutions (containing Escherichia coli OP50) until they reached the adult stage (36–48 hr). Control animals were incubated in M9-containing DMSO solvent to the equivalent concentration as that used in the specific experiment.

HEK293T cells (ATCC #CRL-3216) were cultured in Dulbecco modified Eagle medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM L-glutamine at 37 °C with 5% CO₂. The cells were transfected with the various expression vectors using an established calcium phosphate method^{35 (link)}. To measure the half-life of Flag-tagged Rb mutants, transfected cells were metabolically labeled with [³⁵S] methionine/cysteine for 20 h, washed twice with phosphate-buffered saline (PBS), and chased for the indicated times in regular medium. Cells were harvested, washed with PBS, and lysed with buffer A (20 mM Tris-HCl [pH 7.4], 0.2% [v/v] NP-40, 150 mM NaCl, and 1 mM DTT) containing protease inhibitor cocktail set V (Millipore #539137). The extract was clarified by centrifugation at 20,000 × g for 10 min at 4 °C. PD150606 (Sigma #D5946), MLN4924 (Sigma #505477), carfilzomib (UBPBio #F1300), bortezomib (LC Laboratories #B-1408), MG132 (Enzo #BML-PI102), CIP (New England Biolabs, #M0290), anti-Flag (1:1,000; Sigma #F1804), anti-actin (1:1,000; Sigma #A2066), anti-16E7 (1:1,000; Santa Cruz #sc-51951), and InstantBlue (Expedeon #ISB1L) were purchased from the indicated manufactures. Quantification of bands was performed using ImageJ (National Institutes of Health, version 1.51).

Freshly isolated bone marrow neutrophils (4 × 10⁶ cells/ml) were incubated with 50 nM PMA (Sigma-Aldrich, Stockholm, Sweden) for 3 h at 37 °C in RPMI 1640. In indicated experiments, cells were co-incubated with PAD4 (200 μM, Cl-amidine, EMD Millipore, Billerica, MA), caspase (50 µM, Z-VAD-FMK, R&D Systems) and/or calpain (25 µM, PD150606, Sigma-Aldrich) inhibitors. Supernatants were discharged and fresh media was added to isolate NETs. Residual neutrophils and NETs were removed through extensive pipetting. The mixture was centrifuged at 200 g (5 min) to remove cellular components and NET-containing supernatants were collected. NET-containing supernatants were further co-incubated with PBS or DNAse for 30 min at 37 °C, and then the media with or without NETs were retrieved for further use in vitro experiments.