Methods Reagents and antibodies TMCG was synthesised from catechin by reaction with 3,4,5 trimethoxybenzoyl chloride. DIPY, 4OHT, U0125, and fulvestrant were obtained afatinib mechanism of action from Sigma Aldrich. Antibodies against the following proteins were used B Actin, phospho ATM, phospho Chk2, E2F1, ER, and phospho H2AX. Cell culture and apoptosis assays The MCF 7 and MDA MB 231 human breast cancer cell lines were purchased from the American Type Culture Collection and were routinely authenticated with genotype profiling according to ATCC guidelines. The cells were maintained in the appropriate culture medium supplemented with 10% foetal calf serum and antibiotics. For experiments in hormone deprived conditions cells were maintained for three days in phenol red free DMEM plus 2.
5% dextran charcoal stripped foetal calf serum and then they were treated in the presence or absence of 4OHT. Cell viability was evaluated by a colourimetric assay for mitochondrial function using the 2,3 Bis 2H tetrazolium 5 carboxanilide cell proliferation assay. For this assay, cells were plated in a 96 well plate at a density of 1,000 2,000 cells/well. The compounds were added once at the beginning of each experiment. The Hoechst staining method was used to detect apoptosis. Replicate cultures of 1 105 cells per well were plated in 6 well plates. The cells were subjected to the indicated treatments for 72 h. After changing to fresh medium, the cells were incubated with 5 uL of Hoechst 33342 solution per well at 37 C for 10 min and then observed under a fluorescence micro scope.
Strong fluorescence was observed in the nuclei of apoptotic cells, while weak fluorescence was observed in the non apoptotic cells. The quantification of apoptotic cells was performed by counting the cells in four random fields in each well. PCR analysis mRNA extraction, cDNA synthesis, and conventional and semiquantitative real time PCR were per formed as previously described. The primers were designed using Primer Express version 2. 0 software and synthe sised by Life Technologies. The following primers for hu man ChIP assays A chromatin immunoprecipitation assay was per formed using the Magna ChIP G kit from Millipore according to the manufacturers instructions. Briefly, MDA MB 231 cells were formaldehyde cross linked, and the DNA was sheared by sonication to generate fragments with an average size of 300 to 3,000 bp.
The chromatin was then incubated with anti ER or mouse IgG anti bodies. DNA from lysates prior to immunoprecipitation was used as a positive input control. Dacomitinib After washing, elution, and DNA purification, the DNA solution was used as a template for qRT PCR amplification using specific human primers. Stealth RNA transfection Specific Stealth siRNAs for E2F1 were obtained from Life Technologies and transfected into MDA MB 231 cells using Lipofectamine 2000. The treatments were started 24 h after siRNA transfection.
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