Taqman real-time polymerase Volasertib concentration chain reaction (PCR)-based detection of mature miR-20a was performed by the TaqMan microRNA assays (Ambion, Forest City, CA) as described previously . U6 small RNA was used as an internal control for normalization and quantification of miR-20a expression. All experiments were done in triplicate. Cell proliferation assay Cell proliferation assay was done using cell Titer 96 Aqueous one Solution Cell Proliferation Assay (Promega, Madison, WI) according to the manufacturer’s protocol. Cell cycle analysis HepG2 and SMMC-7721 HCC cells were transfected as described above. After incubated
for 48 h, the cells were typsinized, washed with PBS twice, and then fixed with cold 75% ethanol at 4°C overnight. The fixed cells were centrifuged, resuspended in PBS at 1 × 106
cells/ml and incubated with ribonuclease A and propidium idide (PI) at 37°C for 30 min, then followed by flow cytometric analysis using FL2 histogram of a flow cytometer (FACSort; Becton Dickinson, San Jose, CA). Apoptosis analysis Cells were harvested at the above indicated time points, at least 5 × 105 cells were recovered by centrifugation for evaluation of apoptotic cells with the use of double staining with annexin V–Selleckchem CBL-0137 fluoresein isothiocyanate (annexin V–FITC) and propidium iodide (PI) (BioVision, St Pete Beach, FL) according to the manufacturer’s instructions, followed by flow cytometric selleck chemical analysis with the use of the FL-1 and FL-3 channels of a flow Amino acid cytometer, where apoptotic cells are defined as annexin V + and PI-. Luciferase activity assay For luciferase
reporter assay, HEK293T cells were cultured in 48-well plates and then cotransfected with 10 ng of either pGL3cm-MCL-1-3′UTR-WT or pGL3cm-MCL-1-3′UTR-MUT, 30 pmol of miR-20a precursor or negative control oligonucleotides, and 2 ng of pRL-TK (Promega, Madison, WI). Transfection was done using Oligofectamine (Invitrogen, Carlsbad, CA) according to the manufacturer‘s protocol. Cells were collected 48 h after transfection and analyzed using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI). Experiments were done independently in triplicate. Western bolt analysis Cell lysates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose membrane (Bio-Rad, Hercules, CA) and blocked in phosphate-buffered saline/Tween-20 containing 5% nonfat milk. The membrane was incubared with antibodies for Mcl-1 (Abcam, Cambridge, MA; 1:1000) or GAPDH (Sigma, St. Louis, MO; 1:5000). The antigen-antibody comples was detected using enhanced chemiluminescence (Pierce, Rockford, IL). Immunohistochemical (IHC) staining Paraffin-embedded tissue sections were deparaffinized in xylene and rehydrated in graded series of ethanols followed by heat induced epitope retrieval in citrate buffer (PH 6.0).