Survivor curves selleck bio were estimated by the Kaplan Meier method. Differences in survival were assessed by a log rank test. Statistical analysis of transcription factor assays was done by columnar t test. P values 0. 05 were consid ered statistical significant. Cell lines The human epithelial pancreatic cell line PANC 1 was obtained from ATCC and cultured in Dulbeccos modified Eagles medium supplemented with 10% heat inactivated fetal bovine serum and 1 mM glutamine. RNA interference A nonsilencing siRNA was used as negative control. Cells were transfected with 60 pmol siRNA per well using TransMes senger transfection reagent according to the manufac turers instructions. Efficacy of transfection was checked after 72 h by immunoblotting.

Immunoblotting Total cell lysates from 0,6 105 cells ml were prepared by lysing cells in 30 l p38 buffer for 10 min on ice and centrifugation for 15 min at 14 000 rpm. Supernatant was used to ascertain protein concentration using a BCA Protein Assay Kit. Protein samples were dena turated at 95 C and subsequently separated on a 12% SDS PAGE gel. After transfer to nitrocellulose membrane and blocking with I Block for 20 min the samples were probed with Acetyl H3 antibody, I B antibody, Phospho I B antibody, RelA p65 antibody or antibody against actin over night and washed three times with washing buffer. Incubation with second ary antibody was followed by another wash and subsequent visualisation using CDPstar substrate. Immunofluorescence For immunofluorescence, cells were seeded into 4 well labTEK chamber slides at a density of 0,4 105 cells ml, grown over night and then treated with given concentrations of SAHA or VPA for 72 h.

Stimulation was done by adding 2 l ml IL 1 one hour before fixation. Cells were fixed with ice cold 70% ethanol for 20 min at 20 C and subse quently blocked with serum containing buffer for 30 min. To remove goat serum, chamber slides were washed three times with PBS and probed with primary antibody against RelA p65. Following another wash, cells were incubated with Cy3 conjugated anti mouse anti body and 2,5 l DAPI for 30 min at room temperature and finally visualized by confocal fluorescence micros copy. NF B RelA p65 transcription factor assay For measurement of RelA p65 activity an EZ Detect Tran scription Factor Kit was used, according to the manufacturers instructions.

In brief, 20 g of total protein extract was incubated on a streptavidin coated 96 well plate with bound NF B bioti nylated consensus sequence and detected by specific RelA p65 antibody and secondary HRP conjugated anti body. After adding a chemiluminescent substrate, binding capability of RelA p65 was quantified at a luminometer. To ensure signal Carfilzomib specificity, NF B Competitor Duplex as well as specific RelA p65 siRNA were used as positive negative controls.