| In Vitro | In vitro activity: INH1 reduces the association of Hec1 with kinetochore and decreases global Nek2 protein level in cells. INH1 effectively inhibits the proliferation of human breast cancer lines with GI50 of 10-21 μM. Moreover, INH1 also elicits cell killing activity in part through impairing the Hec1/Nek2 pathway for the spindle checkpoint regulation.
Kinase Assay: Surface plasma resonance (SPR) assays are performed at 22.5°C in HBSD buffer [10 mmol/L HEPES, 150 mmol/L NaCl, 0.1% DMSO (pH 7.5)] on Biacore 3000. 6×His-Hec1 and GST-Nek2 are purified. NTA sensor chip or glutathione-modified CM5 chip are used to capture His-Hec1 and GST-Nek2, respectively. The capture level is about 140 to 180 resonance units (RU) at the flow rate of 5 μL/min. For the binding assay, chips are sequentially treated with compounds (1 or 20 μmol/L) and then proteins (50 μg/mL). Retained RUs are recorded and processed (triplicate experiments).
Cell Assay: Standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays with a 3-d drug treatment procedure are performed to measure the dose-dependent cytotoxicity of INH1 in cultured cells. Triplicate sets are measured and compiled for final data presentation. Cell lines used: MDA-MB-468, SKBR3, T47D, MDA-MB-361, ZR-75-1, HBL100, MDA-MB-435, HS578T, and MCF10A cells. |
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