Following 6 h at 37 C within a humidified 5% CO2 incubator, the cells had been placed in fresh serum containing medium. Cells had been examined just after 48 h within the humidified incubator. Flow cytometry and cell viability To measure cell death, cells had been resuspended in annexin V binding buffer, incubated with five uL of propi dium iodide and subjected to flow cytometry evaluation, making use of a FACS Canto II Flow Cytometer. To allow selective ana lysis in the cells that had incorporated the various hpdODNs, fluorescein labelled hpdODNs had been used. Fluorescein labelled cells were analyzed for PI incor poration or annexin V labelling. A cell death index was established by means of computation of averages. Gel, western blotting Cells were washed in Phosphate Buffered Saline, lysed in sodium dodecyl sulfate sample buffer, 2% SDS, 20% glycerol, 1 mM sodium vanadate, 1 mM dithiothrei tritol and 0.
01% bromophenol blue sonicated and stored at 70 C. Proteins had been separated on SDS polyacrylamide gels and transferred onto nitrocellulose membranes, membranes blocked with dry skimmed milk in Tris Buffered the original source Saline were incubated with antibody overnight at 4 C. Anti phospho STAT1, anti STAT1 and anti STAT3, anti cyclin D1 and anti IRF1 have been employed. Blots had been washed in TBS with Tween, incubated with peroxidase coupled goat anti mouse or goat anti rabbit secondary antibody, washed in TBS T and revealed by chemiluminescence and autora diography. When required, membranes have been stripped with Blot Restore Kit and reprobed with anti tubulin or anti actin antibody to make sure equal loading from the gels. Prestained molecular weight stan dards had been used.
Oligodeoxynucleotide pull down For in cell hpdODN pull down assays, cells have been trans fected together with the biotinylated hpdODNs, as described under transfection, and after that masitinib molecular weight lysed in cell lysis buffer containing salmon sperm DNA. Protein concentration was measured inside the samples. Extracts were recovered on avidin sepharose beads, beads have been incubated for 30 min at 4 C in binding buffer. Soon after washing with binding buffer, complexes had been eluted in SDS sample buffer, separated on SDS Web page, and subjected to immunoblotting using anti STAT1 or anti STAT3 antibodies and processed as above. Immunocytochemistry Cells had been grown at 50 60% confluence in 8 well plates to a density of 105 cells ml. Cells were transfected with fluorescein labelled hpdODNs, incubated, washed in PBS, fixed with 3. 7% formaldehyde for 15 min, permeabilized in 0. 1% Triton X one hundred for 15 min and incubated in 5% FCS 0. 1% Tween PBS for 1 h. Cells have been stained with anti STAT3 or anti STAT1 antibody for two h, then stained with an Alexa fluor 546 labeled secondary antibody for 90 min. Cells, counter stained with four, six diamidino 2 phenylindole, had been mounted onto glass slides with Vectashield.