Supplementary MaterialsTable S1: QPCR primers used in this study. MO, USA). Stock solution of MTX was prepared in 0.1 M NaOH at 1 mg/ml and diluted 1:10 in 0.1 M phosphate buffered saline (PBS) before the use; the pH of the solution was adjusted to 7.4. Lutein was purchased from ChromaDex Inc. (Irvine, CA, USA) and was dissolved in dimethyl sulfoxide NVP-BEZ235 novel inhibtior (DMSO). Reagents for cell culture, including Dulbecco’s modified Eagle’s medium (DMEM), penicillin and streptomycin antibiotic mixture, glutamax, sodium pyruvate and fetal bovine serum were from Invitrogen (Carlsbad, CA, USA). The tetrazolium salt WST-1 (4-[3-(4-Iodophenyl)-2-[4-nitrophenyl]-2H-5-tetrazolio]-1,3-benzene disulfonate) was purchased from Roche Diagnostics (Roche Applied Science, Penzberg, Germany). JC-1 (5,5,6,6-tetrachloro-1,13,3-tetraethylbenzimidazolylcarbocyanine iodide), PI (propidium iodide), and H2DCFDA (6-carboxy-2,7-dichlorodihydrofluorescein diacetate), were purchased from Sigma. Caspase 3 substrate, (Z-DEVD)2-R110 was NVP-BEZ235 novel inhibtior purchased from Bachem (Torrance, CA, USA). Other chemicals unless otherwise stated were obtained from Sigma. Cell culture IEC-6 cells (ATCC#CRL1592) were from American Type Tradition Collection (Rockville, MD). Cells had been cultured in DMEM supplemented with 10% fetal bovine serum (FBS; Invitrogen), 2 mM GlutaMAX-I (Invitrogen), 100 devices/ml penicillin, and 100 g/ml streptomycin (Invitrogen). Cells had been incubated at 37C in 5% CO2. The tradition medium was transformed every 2 times. Cell viability assay The consequences of MTX, only or in conjunction with lutein, in cell viability had been determined by WST-1 reagent, according to the manufacturer’s recommendations. In brief, 4103 cells were seeded on a 96-well plate and allowed to attach for 24 h. the cells were then treated with MTX, lutein, or MTX in the present of lutein. After the indicated time period, cells were incubated for 1 h with 10 l of NVP-BEZ235 novel inhibtior WST-1 reagent and the absorbance was measured using a microplate reader (PowerWave 340, Bio-Tek Instruments, Inc., Winooski, USA) at 450 nm. In the pretreatment experiment, cells were pretreated 2 h or 24 h with lutein prior to MTX exposure and measured of cell viability. Apoptosis assay Apoptosis induction by MTX was assessed by (a) activation of caspase 3 activity; (b) detection of caspase 3 cleavage by Western blot. 1. Caspase 3/7 activity Activation of caspase-3 was assayed using (Z-DEVD)2-R110 substrate as described . Briefly, cells were plated in 96-well plates and allowed to attach by overnight incubation. The cells were then treated with DMSO (control) or the desired concentrations of MTX in the presence or absence of lutein for 24 h. Subsequently, the cells were directly lyzed by adding caspase 3 assay buffer containing (Z-DEVD)2-R110 substrates and incubated at 37C for 1 hour. The fluorescent intensity of proteolytically released fluorophore R110 was then measured using a plate reader (Victor X2; PerkinElmer, Waltham, MA, USA) with an excitation of 485 nm and emission of 535 nm. 2. Detection of caspase 3 cleavage by Western blot Cells subjected to the indicated treatment were harvested and lysed, and the protein concentration was determined by a BCA protein assay (Pierce, Rockford, IL, USA). Protein samples were separated by 14% NVP-BEZ235 novel inhibtior sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA) before probing with antibodies (caspase-3 (active), Epitomics, Burlingame, CA; -actin (as internal control), Sigma). Subsequent immunoblotting procedures were performed using a chemiluminescence procedure (Millipore) per the manufacturer’s instructions. The intensity of immunoreactive bands was determined using GeneTools software (Syngene, Frederick, MD, USA) after scanning the developed films. Results are expressed as mean standard deviation (S.D.) of NVP-BEZ235 novel inhibtior three independent experiments. Detection of mitochondrial membrane potential Changes in the mitochondrial membrane potential in cells were measured by flow cytometry using JC-1 as described . Thirty minutes prior to cytometric analysis, JC-1 is added to the cells to a final concentration of 10 M. Cells are then examined on a FL-1 (530 nm) versus FL-2 (585 nm) dot plot on a FACSCalibur flow cytometer (Becton Dickinson, BD.; NJ. USA). JC-1 has dual emission depending on the state of the mitochondrial membrane potential. It forms aggregates in cells with a high FL-2 fluorescence indicating a normal mitochondrial membrane potential. Loss of the mitochondrial membrane potential results in a reduction in FL-2 and with a concurrent gain in FL-1 fluorescence as the dye NIK shifts from an aggregate to monomeric state. Therefore, the.