Proteins were transferred to nitrocellulose membranes on a semidry electrotransferring unit and incubated with monoclonal rabbit anti-human JMJD2A antibody (Cell Signaling Technology, USA, 1:1000) in Tris-buffered saline containing 0.1% Tween-20 (TBST) and 5% nonfat dry milk overnight at 4°C. After the overnight incubation with the primary antibodies, membranes were washed and incubated with HRP-labelled goat anti-rabbit second antibody (Santa Cruz Biotechnology Inc., USA) in TBST for 2 h. Immunoreactivity was detected with enhanced chemoluminescent

autoradiography (ECL kit, Amersham), according to the manufacturer’s instructions. The membranes were LCZ696 order reprobed with GAPDH (Cell Signaling Technology, USA, 1:1000) after GDC941 striping. The signal intensity of primary antibody binding was quantitatively analyzed with Sigma Scan Pro 5 and was normalized to a loading control, GAPDH [10]. Flow cytometric anlysis (FCM) At 72 h after transfection, cells in different treatment groups were collected with trypsinization, then washed with PBS twice. Cells were fixed in 70% ethanol for 1 h at room temperature. After centrifugation, the cell

pellet was resuspended in PBS (pH 7.4), containing 100 μL RNase A (1 mg/mL) and 400 μL propidium iodide (50 μg/mL). The www.selleckchem.com/products/ly3023414.html cells were incubated for 30 min at room temperature, and DNA content was determined by flow cytometry using a FACScan flow cytometer at 488 nm and the data were input to computer and analyzed by software Light cycle. The experiment

was performed three times in triplicate [11]. Proliferation indexes (PI) was calculated as follows: PI = (S+G2/M)/(G0/G1+S+G2/M)×100%. MTT assay MDA-MB-231 cells were seeded into 96-well plates at a density of 1 × 104 cells per well and incubated in RPMI 1640 medium containing MG-132 in vivo 10% FBS. RPMI 1640 medium containing 10% FBS was replaced by serum-free Opti-MEM 8 h later. These cells were grouped as indicated above (cell transfection). The bulk volume of the transfection compounds was 100 μl per well. Opti-MEM and transfection compounds were replaced by complete medium at 24 h after transfection. After 72 h of incubation, MDA-MB-231 cells were incubated for an additional 4 hours with 20 μl MTT (Sigma Chemical Co., USA, 5 mg/ml). Then the supernatant was removed, and 150 μl DMSO was added. Absorbance at 570 nm (A570) of three groups and DMSO (Sigma Chemical Co., USA) was measured with a microplate reader (Model 550, Bio-Rad, USA) [11]. All experiments were carried out eight times. Actual absorbance = absorbance of the experimental group-absorbance of DMSO. In vitro cell migration and invasion assay At 24 h after transfection, the cells in different groups were treated with trypsin and re-suspended as single-cell solutions. A total of 2 × 105 cells in 0.