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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.