Evodiamine

Evodiamine, the proteasome inhibitor MG132 and the protein synthesis inhibitor cycloheximide (CHX) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The soluble recombinant human TRAIL was purchased from PeproTech, Inc. (Rocky Hill, NJ, USA). Antibodies against Mcl-1, caspase-8, caspase-9, caspase-3, PARP, mTOR, phospho-mTOR (Ser2448), p70 S6 Kinase 1 (S6K1), phospho-S6K1 (Thr389), peroxidase-conjugated secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against DR4, DR5, Bcl-2, Bcl-X_L, Bax, Bad, Bid and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibody against c-FLIP was from Alexis Biochemicals (San Diego, CA, USA). All other reagents were purchased from Sigma-Aldrich unless otherwise specified.

AGS cells and MKN45 cells (1 × 10⁴ per well) were seeded in 96-well tissue culture plates. After 24 h, cells were treated with various concentrations (0.5–20 μM) of evodiamine (≥98% purity; Sigma-Aldrich, St. Louis, MO, USA) dissolved in 100% dimethylsulfoxide (DMSO, ≥99.7% purity; Sigma-Aldrich) for 24 h. The cells were then subjected to water soluble tetrazolium salt (WST) assay by using EZ-Cytox cell viability assay kit (Daeil Lab Service, Seoul, Korea) according to manufacturer’s instruction. The absorbance was measured at 450 nm using NanoQuant Infinite M200 (Tecan, Mannedorf, Switzerland).

LDLR knockout (LDLR−/−) mice were provided by Changzhou Cavens (Cavens, Changzhou, China). To eliminate the effects of sex hormones from the experiment, only male mice were used in the experiment. To induce hypercholesterolemia, 8-week-old LDLR−/− mice were fed a high-fat diet containing 1–1.25% cholesterol (TP28640, Trophic, Nantong, China) for 8 weeks. One group was treated with 10 mg/kg/d by daily gavage evodiamine (Selleck, Houston, TX, USA) for 8 weeks. evodiamine was dissolved in dimethyl sulfoxide (DMSO; Sigma, Abitibi belt, Canada), stored at −20 °C and diluted before use (2% DMSO + 30% PEG400 + saline). The control group was treated with saline. All animal experimental procedures were reviewed and sanctioned by the Animal Ethics Committee of Yangzhou University.

The human colon adenocarcinoma cell lines, HT29, HCT15, and SW480 were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were cultured in Dulbecco’s modified Eagle’s medium or RPMI1640 (both from Corning, Corning, NY, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, USA) and 100 units/ml Anti/anti (Gibco). CSCs were enriched in DMEM/F-12 (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with EGF (Gibco, 20 ng/mL) basic fibroblast growth factor (Gibco, 20 ng/mL), and B27 supplement (Gibco, 1X) on in house prepared ultra low adhesive culture plates, as described previously [35 (link)]. All cells were cultured at 37 °C in a humidified incubator with 5% CO₂. All plastics culture wares were purchased from SPL (Korea). For cell counting, the cells in 96 wells were stained with Hoechst 33342 (Sigma) and imaged using a Cytation™ 3 (BioTek Instruments, Inc. Winooski, VT, USA). The number of CSC spheres with a diameter over 100 μm was counted after taking pictures with Nikon microscope. Evodiamine was purchased from Sigma (USA). The antibodies were acquired from Cell Signaling Technology (Danvers, MA, USA).

For agar dilution test, 10 μL of the bacterial suspension (McFarland 2.0) were placed on the Mueller–Hinton agar (BD Biosciences) supplemented with 10% bovine serum including indicated concentrations of evodiamine (Sigma-Aldrich, St Louis, MO, USA). The bacteria were incubated for 72 h and the minimum inhibitory concentration (MIC) was determined based on the lowest concentration of growth inhibition. For broth dilution test, various concentrations of evodiamine (0.5~40 μM) were treated and the bacteria (McFarland 0.5) were incubated for 72 h. All of the solutions were prepared in such a manner that the final dimethylsulfoxide (DMSO) concentration was the same in all treatments. Final optical density (600 nm) of the bacterial suspension was measured by spectrophotometry.

Evodiamine (#E3531), phorbol 12-myristate 13-acetate (PMA) (#P1585), water soluble cholesterol (#C4951), ApoA1 (#73366), cycloheximide (#C7698), T0901317 (#T2320), and digitonin (#D141) were purchased from Sigma-Aldrich (Vienna, Austria), and pioglitazone (#M35102242) was obtained from Molekula (Munich, Germany). [³H]-cholesterol (#NET139001MC; 1 mCi, 37 MBq) was provided by Perkin Elmer Life Sciences (Vienna, Austria). The tested compounds were dissolved in DMSO, aliquoted and stored at −20 °C until use. An equal amount of DMSO was always tested in each condition in all experiments to assure that the solvent vehicle does not influence the results itself. Primary antibodies against ABCA1 (#NB400-105), ABCG1 (#NB400-132), ABCA12 (NB100-93466) and SR-B1 (#NB400-104) were obtained from Novus Biologicals (Vienna, Austria). The anti-actin antibody (#8691002) was acquired from MP biologicals (Illkirch, France). Primary antibodies against TRFC (#12113) and LDLR (#SC-18823) were bought from Cell Signaling (Austria) and Santa Cruz (Austria), respectively. HRP-linked anti-rabbit IgG secondary antibody (#7074S) was purchased from New England Biolabs (UK), and horseradish peroxidase conjugated goat anti-mouse secondary antibody (#12-349) from Upstate (Millipore, Vienna, Austria). All antibodies were used in a dilution of 1:500.