Then cellular viability was evaluated. Plasmids pIRES/hygro and pIRES/hygro-full CLU expressing vectors have been previously described . Vector expressing short hairpin RNA against CLU RNA (CLU-shRNA; ver.3) was purchased from Upstate Biotechnology (Lake Placid, NY, USA). Generation of cell lines stably expressing
s-CLU OVK-18 cells were cultured to 50% confluence. Plasmid DNA transfection was done using Effectine (Qiagen) according to the manufacturer’s instructions. pIRES-hygro or pIRES-CLU-hygro-transfected OVK18 cells were selected in hygromycin (50 μg/ml; Sigma). Selected colonies were screened by immunoblotting to identify stable clones expressing s-CLU. Cell viability assay Cell viability was evaluated using cell counting kit (CCK-8) (Dojindo, Kumamoto, Japan). Briefly, transfected cells were pre-cultured in 96-well Lonafarnib cost plate (3,000 cells/well) for 24 h. Seventy two hours after TX treatment at the indicated doses, culture media were replaced by the WST-8 reagent. Reduced WST-8 by the cellular dehydrogenases
turns into orange formazan. Produced formazan is directly proportional to living cells. Absorbance was measured at 450 nm by microplate reader equipped by computer (NEC, Tokyo, Japan). Flow cytometry analysis Following TX treatment, cells were trypsinized, washed twice in phosphate-buffered saline (PBS) and cell cycle phases were analyzed. Briefly, cells were fixed at 4°C overnight in 70% ethanol. After washing with Ca2+-Mg2+-free Dulbecco’s PBS, cells were treated with 0.1 μg/ml RNase (Type I-A, Sigma), stained with 100 μg/ml propidium iodide (PI; Sigma) for 20 min, find more filtered and kept on ice until measurement. Cells were acquired by the FACS calibrator (BD, Bioscience) and then analyzed using the ModFit FHPI chemical structure software (Verity software; ME, USA). Cell fractions with a DNA content lower check than Go/G1, the sub-G0/G1 peak, were quantified and considered
a marker of the number of apoptotic cells. Annexin V staining After harvesting and washing as described above, the cells were stained directly with PI at final concentration of 10 μg/ml and 2% Annexin-V Flous (Roche, Basel, Swizerland) in incubation buffer (10 mM Hepes/NaOH, pH 7.4, 140 mM NaCl, 5 mM CaCl2) for 10 minutes. Cells were acquired with the FACS calibrator (BD) after setting the instrument with controls (non-treated, stained cells) after two washes in PBS. In this experiment, both cells with early apoptotic signals, stained with annexin V, and cells with late death signals, stained with PI, are all considered and quantified. Apoptotic cells were analyzed using the CellQuest software. Western blotting Cell lysates were obtained by resuspending cells in RIPA buffer (10 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% Nadeoxycholate (Kanto Chemical, Tokyo, Japan) and 5 mM EDTA) supplemented with protease inhibitors cocktail (Sigma, USA).