After 20 min shaking at room temperature lactate dehydrogenase activity was determined by change in absorbance at 490 nm. All drug concentrations were tested at least in triplicate wells and the assays were re peated independently three times. TUNEL assay TUNEL was carried out directly using an In Situ Cell Death De tection Kit, AP according to the manufacturers instructions. Briefly, after 48 h treatment by 2 uM TAM, 200 uM tranilast or a combin ation two, the cells were fixed Inhibitors,Modulators,Libraries by adding 4% paraformalde hyde for 30 min. The fixed cells were washed in PBS, permeabilized with 0. 1% Triton X 100 for 5 min on ice, and then incubated with 50 ul of terminal deoxynucleotidyl transferase end labeling solution for 60 min at 37 C in a humidified chamber in the dark.
Then, cells were coun terstained in PI staining solution for 4 min at Inhibitors,Modulators,Libraries room tem perature in the dark. The percentage of positively stained cells per total number of cells was counted under a fluores cence microscope at a magnification of 40�� in five random fields and averaged. Acridine orange/ethidium bromide staining MCF 7 or MDA MB 231 were plated in 24 well plates and incubated overnight in a humidified 5% CO2 incubator at 37 C for 24 h. At that time, cells treated with 2 uM TAM, 200 uM tranilast or a combin ation two and incubated for 48 h. After that, cells har vested and stained with AO/EB dye mix on a clean microscope slide. The live, apoptotic and necrotic cells were observed under the fluorescent microscope at a mag nification of 40��. Experiments were repeated for twice.
DNA gel electrophoresis The MCF 7 and MDA MB 231 cells were grown in ab sence or presence Inhibitors,Modulators,Libraries of 2 uM TAM, 200 uM tranilast and combination of both for 48 h. Cellular DNA was then extracted from each cell line. The cells were lysed with 1% SDS in TE buffer and digested with proteinase K for 4 h at 56 C. The samples were extracted with phenol and chloro form and the DNA was precipitated with a 1/10 volume of 3 M sodium acetate and an equal volume of ethanol, pelleted at 13,000 g and resuspended in TE buffer and 10 mg/ml of DNase free RNase for 30 min at 37 C. Fi nally, extracted genomic DNAs was loaded and fractioned on 2% agarose gels. gels were stained with Inhibitors,Modulators,Libraries ethidium brom ide and photographed. When DNA extracted from apop totic cells is subjected to gel electrophoresis, a typical internucleosomal ladder of DNA fragments is produced.
Real time quantitative PCR analysis Total Inhibitors,Modulators,Libraries cellular RNAs were extracted from control or drug treated cell pellets, 48 h after treatment with 2 uM TAM, 200 uM tranilast and combination both, using RNeasy Mini kit in accordance with the manu facturer s protocol. First strand cDNA was synthesized using QuantiTect selleckchem Reverse Transcription Kit. Numbers of cDNA copies were calculated from the ab sorbance at 260 nm.
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