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note
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50 values are determined using a regression analysis of the concentration/inhibition data.
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note
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2 for 72 h. Then, 10 μL of alamarBlue™ was added, and cells were incubated at 37 °C for 3 h. The fluorescence of the wells was measured on a fluorometric reader with excitation set at 530 nm and emission detection set at 590 nm, and the percentage of cell growth was calculated from the fluorescence readings.
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26
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note
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5) were treated for 14 h with samples at the indicated concentrations in 10% FBS-supplemented DMEM then collected and extracted with SDS buffer. Protein concentrations of the lysates were determined using a Bradford protein assay kit (Bio-Rad Laboratories); equivalent amounts of proteins from each lysate were resolved in 15% SDS-polyacrylamide gel and then transferred onto nitrocellulose membranes (Bio-Rad Laboratories). After blocking for 30 min with Tris-buffered saline (TBS) containing 3% skim milk, the transblotted membrane was incubated overnight at 4 °C with hyperacetylated histone H4 antibody (Upstate Biotechnology) (1:2000 dilution) or β-actin antibody (Abcam) (1:1000 dilution) in TBS containing 3% skim milk. After probing with the primary antibody, the membrane was washed twice with water, then incubated with goat anti-rabbit or anti-mouse IgG-horseradish peroxidase conjugates (diluted 1:5000) for 2 h at room temperature, and further washed twice with water. The immunoblots were visualized by enhanced chemiluminescence.
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