Protease inhibitor

CE81T/VHG cells were seeded in 100 mm culture dishes for incubation overnight, and then treated with 70 μg/mL JCo extract for 0, 6, 12, 24, or 48 hr. Cells were lysed with ice‐cold RIPA buffer containing protease inhibitor (BIO BASIC INC.) and phosphatase inhibitor (Bionovas) and incubated at 4°C for 30 min. The resulting lysates were centrifuged at 12,000 × g for 30 min at 4°C, and the protein concentration was determined using a bicinchoninic acid protein assay kit (Pierce, Rockford, IL, USA). Cell extracts (20 μg protein/lane) were separated using 8%–12.5% SDS‐PAGE gel electrophoresis and transferred to polyvinylidene difluoride membranes (FluoroTrans, PALL). The membrane was blocked with 10% skim milk and incubated with primary antibodies or β‐actin (iReal Biotechnology) in 1% Bovine serum albumin (in 1× TBS with 0.1% tween‐20) overnight, followed by incubation with biotin conjugated secondary antibodies for 2 hr. Finally, membranes were incubated with horseradish peroxidase‐conjugated streptavidin (Dianova) for 1 hr and proteins were detected using an ECL kit (T‐Pro Biotechnology) and chemiluminescence imaging analyzer (GE LAS‐4000, GE healthcare Life. Sciences). Protein bands were quantified using the software ImageJ 1.47t and normalized to the mean values of the untreated control and β‐actin.

Lipo-PEI-PEG-complex (LPPC) was provided by the lab of Dr. Kuang-Wen Liao, National Yang Ming Chiao Tung University, Hsinchu, Taiwan. Anticancer peptides (ACP) were synthesized and purchased from AngeneBiotech (Taipei, Taiwan). Phenol, chloroform, RIPA lysis buffer and protease inhibitor were purchased from Bio Basic Inc. (Toronto, Canada). MTT, propidium iodide (PI), and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich Chemicals Co. (St. Louis, MO, USA). Pierce™ ECL Western Blotting Substrate was purchased from Thermo (Thermo Fisher Scientific Inc., Waltham, MA, USA). Myco5A medium, M199 medium, EMEM medium, DMEM medium, RPMI-1640 medium, fetal bovine serum (FBS), PBS buffer solution, sodium pyruvate (100 mM), and 0.05% trypsin-EDTA were purchased from Gibco (Grand Island, NY, USA).