Abt 263

Protein was extracted from cells pretreated in RIPA buffer with or without ABT-263 (Thermo Fisher Scientific). The protein concentration was calculated using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Protein samples (10 µg) were then loaded onto Mini-PROTEAN TGX Precast Gels (BIO-RAD) and separated by electrophoresis. The separated proteins were transferred to Trans-Blot Turbo Mini PVDF membranes (BIO-RAD), and the membranes were blocked with 5% skim milk for 1 h. The membranes were then incubated with the primary antibodies for BCL-2 (1:1000; Cell Signaling Technology) and β-actin (1:1000; Cell Signaling Technology) overnight at 4 °C, washed with Tris-buffered saline containing 0.05% Tween-20, and then incubated with the secondary antibodies conjugated with horseradish peroxidase (1:5000; Abcam) for 1 h at room temperature. SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific) was then applied to the blot. Chemiluminescence was detected by ChemiDoc XRS + system (BIO-RAD) and analyzed by Image Lab 6.1 (BIO-RAD). The integrated intensity of BCL-2 was normalized to that of β-actin.

Hypoxia green reagent; pHrodo Green E. coli; DAF-FM DA; JSH-23; QNZ; ABT-263; and ABT-737 were purchased from Thermo Fisher Scientific (Waltham, MA, USA). The fetal bovine serum was from Gibco (Gibco, Sigma Aldrich Company Ltd., Waltham, MA, USA). Intracellular staining permeabilization wash buffer; Human TruStain FcX (Fc Receptor Blocking Solution); PE anti-human Ki-67; isotype control antibodies PE Mouse IgG1 k isotype Ctrl; APC Mouse IgG1 k isotype Ctrl; FITC Mouse IgG1 k isotype Ctrl; PE Mouse IgG2a k isotype; PE Mouse IgG2b k isotype and antibodies APC anti-human CD11b; PE anti-human CD284; PE anti-human CD36; PE anti-human CD33; PE anti-human HLA-DR; PE anti-human HIF-1α were from BioLegend (San Diego, CA, USA). MycoFluor™ Mycoplasma Detection Kit was from Molecular Probes Inc. (Eugene, Oregon, USA). FITC anti-human CD68 was from BD Bioscience (Franklin Lakes, NJ, USA). Culture medium RPMI 1640; 2-mercaptoethanol; PBS; Calcein AM; Bisbenzimide Hoechst 33342 (H33342); propidium iodide (PI), resazurin; LPS of E. coli O111: B4; antibodies FITC anti-human CD11c; FITC anti-human CD14; FITC anti-human CD54; NF-kB activation inhibitor IV, and other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).