Goat anti mouse or goat anti rabbit secondary antibodies

K562-WT1-siRNA-GFP⁺ cells and K562-C-siRNA-GFP⁺ cells were lysed by the CelLytic M reagent (Sigma) for 15 minutes. Protein lysate was centrifuged at 12,000 rpm for 15 minutes, and the protein concentrations were determined using the Bradford protein assay (Sigma). Twenty micrograms of protein was separated on SDS-PAGE and then transferred to a PVDF membrane (Whatman) using blotting buffer for 1 hour. Then, the membrane was blocked for 1 hour and probed with specific primary antibodies (1 : 100 of polyclonal anti-WT1 antibody (Santa Cruz, C19) or 1 : 1000 of polyclonal anti-actin antibody (Santa Cruz, H196) or 1 : 1000 of anti-caspase-7 antibody (Sigma, C7724) in 1% skim milk at room temperature for 2 hours. Consequently, the membrane was detected with the immunocomplexes using horseradish peroxidase conjugated with either an appropriated dilution of goat anti-mouse or goat anti-rabbit secondary antibodies (Santa Cruz). The immunocomplex was detected with SuperSignal Pico Chemiluminescent Substrate for 5 minutes followed by film exposure.

Cells (1 × 10⁶ cells in 10 ml medium) were seeded in 10-cm dishes. After the NSCLC cells were treated with DMSO or TBPT, they were washed and collected. Cell lysates were prepared conventionally in RIPA lysis buffer. The protein concentrations were determined using the BCA protein assay. Equal amounts of total protein were loaded onto SDS-PAGE gels (8–10%) and transferred onto PVDF membranes. After the membranes were blocked with 5% nonfat milk for 1 h, they were blotted with primary antibodies against P-gp, β-actin, full-length caspase 3 (Santa Cruz Biotechnology, Dallas, TX, USA), caspase 8 (BD Biosciences, San Jose, CA, USA), p21, caspase 9, cleaved caspase 3 and PARP (CST) at 1 : 500–1 : 1000 dilution overnight at 4 °C. Then, the membranes were incubated with goat anti-mouse or goat anti-rabbit secondary antibodies (1 : 5000, Santa Cruz) at RT for 1 h. An enhanced chemiluminescence western blot system (Millipore) was used to detect the immunoreactive bands. Intensity of the blot was determined using the ImageJ software (NIH, Bethesda, MD, USA).

Hor (high-performance liquid chromatography-grade ≥95%) was purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Dimethyl sulfoxide and LPS were purchased from Sigma Aldrich (St. Louis, MO, United States). Phosphate-buffered saline and 0.05% pancreatic enzyme were purchased from Solarbio Technology (Beijing, China). Penicillin and streptomycin, fetal bovine serum, and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from Hyclone Laboratories (Logan, UT, United States). Primary antibodies [COX-2, iNOS, protein kinase B (AKT), p-AKT, NF-κB-p65, NF-κB-p-p65, inhibitor of NF-κB (IκB), p-IκB, p38, p-p38, c-Jun N-terminal kinase (JNK)1/2, p-JNK1/2, extracellular signal-regulated kinase (ERK)1/2, and p-ERK1/2] were purchased from Cell Signaling Technology (Danvers, MA, United States), and primary antibodies against β-actin and goat anti-mouse or goat anti-rabbit secondary antibodies were obtained from Santa Cruz Biotechnology (Dallas, TX, United States).