Fig. 2.

Basal levels of NFκB do not correlate with gemcitabine sensitivity. A, NFκB nuclear binding was analyzed in human pancreatic cancer cell lines. Nuclear extracts from six pancreatic cancer cell lines and human pancreatic duct epithelial cells were prepared and EMSA using a labeled oligonucleotide (Oligo) containing a consensus κ B binding site was done. TBP was used as a loading control for quality and quantity of cell extracts. Negative control, ³²P-labeled NFκB Oligo without HeLa nuclear extract; positive control, HeLa nuclear extract and ³²P-labeled NFκB Oligo; specific competitor, HeLa nuclear extract with ³²P-labeled NFκB Oligo plus unlabeled NFκB Oligo; nonspecific competitor, HeLa nuclear extract with ³²P-labeled NFκB Oligo plus unlabeled Oct1 Oligo. B, NFκB transcriptional activity was estimated using an NFκB reporter assay. Pancreatic cancer cells were coinfected with an NFκB reporter expressing firefly luciferase and a control lentivirus expressing renilla luciferase, and stable populations were developed. Basal NFκB transcriptional activity determined as a ratio of firefly and Renilla luciferase signals and presented as a comparison with the level measured in human pancreatic duct epithelial cells. As a positive control, all cell lines were also challenged with TNF-α (10 ng/mL) for 6 h and the level of NFκB activity was determined. Data shown are means ± SE for three independent experiments.