3 4 5 dimethythiazol 2 yl 2 5 diphenyl tetrazolium bromide mtt

The human colorectal cancer cell line HCT15 was obtained from the American Type
Culture Collection (Manassas, VA). Cells were grown in a monolayer culture in
DMEM (Sigma) supplemented with 10% fetal bovine serum (Sigma) and 1%
streptomycin/penicillin, and were maintained at 37°C in a humidified
atmosphere consisting of 5 % CO₂ and 95% air. Cells were regularly
tested for mycoplasma contamination by treating 5 ug/mL of Plasmocin
(InvivoGen). NVP-BEZ235 was purchased from LC laboratories (Woburn, MA). CQ,
N-acetylcysteine (NAC), trolox, acridine orange,
3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and
Hoechst33342 were all purchased from Sigma and Image-iT LIVE Green Reactive
Oxygen Species Detection Kit was from Molecular Probes.

Cultured C. militaris was purchased from Honghao Biological Company of Jiangmen (Guangdong, China). CMF was isolated, identified, and purified as per our previous report.[11 (link)] CMF stock solution was prepared into 1000 μg/ml concentration in 1640 complete medium and stored at 4°C. The antibodies for p-c-Jun, c-Jun, p-ERK, ERK, p-p38, p38, p-JNK, JNK, GAPDH, PARP, p-p53, cyclin B1, cdc2, cdc25c, caspase-3, and caspase-9 were obtained from Cell Signaling Technology, Inc. (Boston, MA, USA). Antibodies for Bax, Bcl-2, and cytochrome c were purchased from Abcam Ltd. (Cambridge, UK). SB203580, SP600125, and 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were purchased from Sigma-Aldrich, Inc. (Saint Louis, MO, USA). Roswell Park Memorial Institute (RPMI)-1640 medium and fetal bovine serum (FBS) were purchased from Gibco/Life Technologies Inc. (Carlsbad, CA, USA).

α-mangostin (chemical structure shown in Fig. 1) was purchased from Sigma-Aldrich (St. Louis, MO, USA), dissolved in dimethyl sulfoxide (DMSO) and stored at −20°C. RPMI-1640 medium, penicillin-streptomycin, trypsin-EDTA and fetal bovine serum (FBS) were purchased from HyClone Laboratories, Inc. (Logan, UT, USA). 3-(4,5-Dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and DMSO were obtained from Sigma-Aldrich. Cell lysis buffer and 4′,6-diamidino-2-phenylindole (DAPI) were purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). The fluorescein isothiocyanate (FITC)-conjugated Annexin V Apoptosis Detection kit was purchased from BD Biosciences (San Diego, CA, USA). Anti-β-actin (#4967), anti-Bax (#2772), anti-Bcl-2 (#2876), anti-caspase-3 (#9662), anti-cleaved caspase-3 (#9661), anti-caspase-9 (#9502), anti-poly(ADP-ribose) polymerase (PARP; #9542), anti-ERK1/2 (#9102), anti-phosphorylated (p)-ERK1/2 (#4376), anti-p38 (#9212), anti-p-p38 (#4631), anti-c-myc (#9027), anti-Ki-67 (#9027) and goat anti-rabbit horseradish peroxidase (HRP)-conjugated (#7074) antibodies were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). The DeadEnd™ fluorometric terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay kit was purchased from Promega Corp. (Madison, WI, USA).

The human colorectal cancer cell line HCT15 was obtainedfrom the American Type Culture Collection (Manassas, VA). Cells were grown in a monolayer culture in DMEM (Sigma) supplemented with 10% fetal bovine serum (Sigma) and 1% streptomycin/penicillin, and were maintained at 37°C in a humidified atmosphere consisting of 5% CO2 and 95% air. Cells were regularly tested for mycoplasma contamination by treating 5 μg/mL of Plasmocin (InvivoGen). BEZ235 was purchased from LC laboratories (Woburn, MA). CQ, acridine orange, 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were all purchased from Sigma.