Annexin-V assay Surface exposure of phosphatidylserine in apoptotic cells was measured by Guava Nexin Kit according to the manufacturer’s protocol (Guava Technologies, Hayward, California, USA). Annexin-PE fluorescence was analyzed by cytosoft software (Guava Technologies, Hayward, California, USA). Wherever indicated HEK293T cells were pre-treated with BOC-D-FMK (broad caspase inhibitor, 300 mM, Sigma, St. Louis, Missouri, USA) for 30 min before transfection. Measurement of intracellular ROS generation, mitochondrial membrane potential (Dym) and cytochrome-c release Mitochondrial membrane potential was measured using DiOC6 (3,39-dihexyloxacarbocyanine iodide) dye and ROS production was measured using DCFH-DA (29,79-dichlorfluorescein-diace- tate) by flow cytometry as described before [29]. In brief, untransfected and p(23a,27a,24-2) transfected HEK293T cells were washed with PBS and DCFH-DA (5 mM) or fluorochrome DiOC6 (40 nM) was added 30 min prior to harvesting. Fluores- cence intensity was monitored using flow cytometer (Guava Technologies, Hayward, California, USA). A minimum of 10,000 events were counted. Cytochrome-c release was observed using Cell Fractionation Kit from BD Biosciences [30]. In brief, untransfected and p(23a,27a,24-2) transfected HEK293T cells were trypsinized, harvested, washed once with PBS and resuspended in 100 ml of lysis buffer. The cells were then homogenized and centrifuged to separate mitochondrial pellet and cytosolic fraction. Cytochrome-c release was analyzed in cytosolic and mitochondrial fraction by immunoblotting. Cox IV was used to check the purity of cytosolic and mitochondrial fraction. Equal loading was confirmed using b- actin. Protein preparation and western blot analysis Untransfected and p(23a,27a,24-2) transfected HEK293T cells were trypsinized and cell pellets were lysed with modified RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP40, 0.25% Na deoxycholate, 1 mg/ml aprotinin, 1 mg/ml leupeptin, 1 mg/ml pepstatin, 1 mM PMSF, 1 mM sodium orthovanadate, and 1 mM sodium fluoride) and kept in ice for 30 min. Lysate was centrifuged at 12000 rpm for 30 min and supernatant was collected. Protein concentration was determined using the Bio-Rad protein assay kit. Equal amounts of sample lysates (40 mg) were separated by 12% sodium dodecyl sulphate - polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membrane (mdi, Advanced Microdevices, India). The membrane was blocked with 3% skim milk in Tris buffered saline (20 mM Tris, 150 mM NaCl, pH 7.4) with 0.1% Tween-20 for 1 h and then incubated with primary antibody (FADD/caspase 8/ caspase 3/PARP/Bid/Bcl-2/Bax) in 1% skim milk for 2 h followed by incubation with appropriate secondary antibody (anti-mouse ALP linked or/anti-rabbit HRP linked) for 1 h. Blots were developed using enzyme based chemiluminescence assays (Alkaline phosphatase or Horse-radish peroxidase) using NBT- BCIP or DAB as substrate. Equal loading of protein was confirmed using b-actin antibody or GAPDH antibody. All experiments were repeated at least three times; representative results are presented [31]. Quantitiative and Statistical analysis of western blot data Measurement of signal intensity on PVDF membranes after western blotting with various antibodies was performed using miRNA Cluster Induce Apoptosis AlphaImager 3400 (Alpha InnoTech Corporation, San Leandro, California). All data were expressed as integrated density values (IDV). For FADD, Caspase 3, Caspase 8, Bcl-2, Bax, Bid, PARP the IDV values were calculated as the density values of the specific protein bands/b actin or GAPDH density values. All figures showing quantitative analysis include data from atleast three independent experiments. Statistical analysis was performed with student’s two tailed t-test using SPSS (windows version 7.5); values of p#0.05 were considered statistically significant. Northern Blotting Total RNA was extracted with the Trizol reagent as per the manufacturer’s instructions. For Northern blotting, the total RNA (40 mg) was run on a 15% polyacrylamide-urea gel, transferred to a Hybond N+ membrane using semidry apparatus (BioRad, Hercules, CA, USA) and UV-crosslinked (Stratalinker). The blot was probed with a RNA probe using miRVana probe construction kit (Ambion Austin, TX, USA). The blot was scanned in the phosphorimager after overnight exposure. The normalization of the result was done by stripping the blot in 0.01% SDS at 80uC for 2 hours and probing it for U6 expression. Probe sequence for 27a and U6 Probe 27a: TTC ACA GTG GCT AAG TTC CGC CCT GTC TC Probe U6: TGC TAA TCT TCT CTG TAT CG Luciferase assay Twenty-four hours before transfection, HEK293T cells were plated in 6-well plate. pMIR-REPORT miRNA Expression Reporter Vector containing 39UTR region of the FADD and/or p-Silencer 4.1 vector expressing hsa-miR-23a,27a,24-2 (p(23a,27a,24-2)) were transfected into HEK293T cells using lipofectamine 2000 (Invitrogen, CA, USA) as described by the manufacturer. pCDNA 3.1/anti-miR-23a/anti-miR-27a/anti- miR-24-2 (40 nm/80 nm Ambion, Austin, TX, USA) were also used wherever indicated. Luciferase activity was measured using Luciferase Kit Assay System (Promega, Madison, WI, USA) and readings were taken on the luminometer (Berthold AutoLumat LB953 rack luminometer). Luciferase activity was then normalized by b-galactosidase activity for transfection in each well. Reporter assay The pNF-kB-luc reporter plasmid containing direct repeats of the transcription recognition sequence for NF- kB (path detect NF- kB Cis-Reporting system) was a kind gift from Zdenek Dvorak. For reporter assay, untransfected and p(23a,27a,24-2) transfected HEK293T cells were taken and following 8 h incubation period, cells were treated with TNF-a (10 ng/ml) for 16 h. At the end of treatment, cells were lysed and luciferase activity was measured and standardized per milligram of protein [32]. Immunostaining and confocal microscopy Cells were seeded on coverslips in six-well plate (Tarsons) at 1.66105 to 26105 per well for 24 h prior to transfection. Transfection was done with p(23a,27a,24-2) using lipofectamine 2000 as described by the manufacturer. Cells were rinsed with PBS and fixed with ice-cold acetone: methanol mix (1:1) for 10 min at 220uC. They were rinsed again and permeabilized with 0.5% Triton X-100 in PBS for 15 min at room temperature. This was followed by washing with PBS and consequently blocking for 45 min in 1% bovine serum albumin in PBS at room temperature, PLoS ONE | www.plosone.org 3 June 2009 | Volume 4 | Issue 6 | e5848