Viable, apoptotic and necrotic cells were counted in 10 different fields under the 200�� vision in each well in three independent experiments by two persons and the average result was compared to the mock selleck chemical H89 control. Apoptotic cell numbers from different treatments were compared by being normalized to their viable cell numbers. Flow cytometry analysis of cell cycle HepB3 cells were selected to test the effect of miR 221 on cell cycle. Cells were plated into 6 well culture plates and treated with miR 221 inhibitor, mimic and their negative controls for 96h. Cells were collected with trypsin, then washed once with 4 C PBS, and fixed in cold 75% ethanol at 4 C. Cells were then washed once again with 4 C PBS and re suspended with PBS, then stained with 50 mg ml PI and 100 mg ml RNase A solution for 20 min at 37 C in dark.
Stained cells were subjected to analysis immediately by flow cytometry. The proportion of cells in each phase of the cell cycle was determined by a BDFACScan for Quantitative Cell Analysis. Caspase 3 7 activity detection Caspase 3 7 activity was measured using a synthetic rhodamine labeled caspase 3 7 substrate performed immediately after the detection of cell viability on the same wells, according to the instructions of the manufacturer. After incubation at room temperature for 60min, the fluor escence of each well was measured, using a FL600 fluorescence plate reader. Caspase 3 7 activity is expressed as fluorescence of treated sample mock control��100. Flow cytometry analysis of apoptosis HepB3 cells were also selected further to confirm the effect of miR 221 on apoptosis, using 7 Amino Actinomycin APC Annexin V with flow cytometry.
Cells were prepared as above and the procedure was according to the manufacturer. This assay allows to identify early apoptotic cells and late apoptosis or already dead. Statistical analysis SPSS19. 0 was used for statistical ana lysis. Results were representative of three independent experiments unless stated otherwise. Values were presented as the mean standard deviation. One way Analysis of Variance test and Students paired t test were used to analyze significance between groups. The Least Significant Difference method of multiple compa risons with parental and control group was applied when the probability for ANOVA was statistically significant. Statistical significance was determined at a P 0.
05 level. Background Up to 40% of patients with early stage breast cancer have disseminated tumors cells in the bone marrow. Fur thermore, bone is the most common site for breast cancer metastasis with 50% of metastatic breast cancer patients presenting with bone metastasis. Increased bone resorption is becoming increasingly recognized as a risk factor for development of metastatic tumor in the bone.
After transfection, intermediate samples at 24, 48 and 72 hrs were collected and analyzed by different assays. Western blot analysis After transfection with miR 221 inhibitor, miR 221 mimic and different controls, the cells were washed with PBS and lysed in a buffer containing Tris HCL 20mM, NaCl 150mM, EDTA 1mM, TRITON X 1%, Na pyrophosphate 2. H89 IC50 5mM, Sodium orthovanadate 1mM, Leupeptin 1ug ml, pro tease inhibitor cocktails 1% and phosphotase inhibitor cocktails 1%. The lysates were centrifuged at 12,000 g for 10 min at 4 C and boiled for 5 min. The protein concentration of the lysate was detected by the Bio Rad Bradford protein assay and 25ug of denatured protein was subjected to SDS PAGE with a loading buffer containing 80mM Tris HCl, 5% SDS,10% glycerol, 5mM EDTA, 5% 2 Mercapto Ethanol, 0.
2% Bromophenolblue and 1mM phenylmethylsulfonyl fluoride The separated proteins were transferred to PVDF membranes for 2 hrs at 100 mA. The membrane was incubated with a p27 Kip1 Mouse Monoclonal Antibody, p57 Kip2 Rabbit Polyclonal Antibody or a B actin antibody. Primary antibodies were detected with an HRP conjugated secondary anti body and finally the membranes were subjected to chemiluminescence detection assay. Experiments were repeated in triplicate. Cell viability Cell viability was assessed using a fluorimetric detection of resorufin. The protocol was as follows miR 221 inhibitor, miR 221 mimic and their negative controls were transfected to 96 well plates and incubated at 37 C for up to 96 hrs. The procedure was according to the manufacturer.
Fluorimetry was using an FL600 fluorescence plate reader. Fluorescence data are expressed as the fluorescence of treated sample mock control 100. Cell proliferation To further confirm the data from the above cell viability assay, cell proliferation was detected by a colorimetric tetra zolium assay. The treatments and controls were as mentioned above. After transfections, addition of 20 ul of MTS reagent to each well, the plates were incubated for 2 hrs at 37 C in a humidified 5% CO2 atmosphere, and the absorbance at 490 nm was recorded using a 96 well microplate reader. The results were the mean of six wells and expressed as the ratio of the absorbance of different transfections absorbance of mock control 100.
Fluorescent microscopy evaluation of cell apoptosis and morphology Besides the CellTiter Blue cell viability assay and MTS assay, cell growth was also monitored with Hoechst 33342 and propidium iodide double fluorescent chromatin staining. With this assay, the effects of miR 221 inhibitor and mimic on apoptosis and nuclear morphology in the HCC cells could also be assessed. In brief, after different transfections, cells were washed with ice cold PBS and stained 15min with Hoechst 33342 and PI, and observed under an advanced fluorescence microscope. Apoptosis and nuclear morphology were identified by condensation of nuclear chromatin and its fragmentation.