Article Figures & Data
Figures
- Fig. 1.
Immunohistochemical detection of activated RelA in paired normal and tumor pancreatic tissue. Twenty-four paired normal and tumor pancreatic tissue samples were subjected to immunohistochemical analysis using a RelA monoclonal antibody specific for the activated RelA protein. The subsequent analysis was carried out as described previously (36) . A, normal pancreas tissues; B, pancreatic adenocarcinoma; and C, pancreatic adenocarcinoma with the control peptide were probed with the anti-RelA antibody specific for activated RelA. Representative fields are shown above.
- Fig. 2.
RelA activity in nuclear extracts isolated from paired normal and tumor pancreatic tissue samples. A, nuclear extracts (50 μg) were used in EMSA to determine the RelA-DNA binding activity in paired normal (N) and tumor (T) pancreatic tissues (paired Lanes 1–8), using the HIV κB oligonucleotides as probes. B, the nuclear extracts from the tumors (paired Lanes 1 and 2) were used in competition with a 50-fold excess of unlabeled wild-type or mutant κB oligonucleotides and in supershift with anti-RelA antibody in the absence or presence of the control peptide. Arrow, supershift complex. In C, cytoplasmic extracts (25 μg) were used in Western blot analysis with IκBα antibody specific for the NH2 terminus (amino acids 1–56) of IκBα protein. The subsequent Western blot analysis was carried out with the ECL Western blotting kit described in “Materials and Methods” (10) .
- Fig. 3.
RelA activity in tumorigenic and nontumorigenic pancreatic cell lines. Control cells were treated with TPA (50 μg/ml) or with TNF-α (5 ng/ml). In A and B, nuclear extracts (10 μg) were subjected to EMSA to determine the RelA-DNA binding activity in tumorigenic human and SGH pancreatic cell lines and nontumorigenic SGH pancreatic cell lines as indicated. In C, nuclear extracts from MDAPanc-28 and control Jurkat cells were subjected to EMSA for competition with a 50-fold excess of unlabeled wild-type or mutant κB oligonucleotides and for supershift using anti-RelA antibody with or without control peptide. The HIV κB oligonucleotides were used as probes. Arrow, supershift complex. D, expression of IκBα in human pancreatic tumor cell lines. Total RNA or cytoplasmic extracts were isolated from the cell lines as indicated with or without treatment with TPA (50 μg/ml) or TNF-α (5 μg/ml). Total RNA (25 μg) was used in Northern blot analysis. The blots were hybridized with a human IκBα (MAD3) cDNA probe, exposed, stripped, and rehybridized with the cDNA probe for glyceraldehyde-3-phosphate dehydrogenase (data not shown).
- Fig. 4.
A, inhibition of RelA-DNA binding activity by curcumin. MDAPanc-28 cells were grown to 80% confluence and treated with various concentrations of curcumin for 6 h as indicated. Nuclear extracts (10 μg) were used in EMSA to determine the RelA-DNA binding activity in the cell lines treated with or without curcumin. B, inhibition of constitutive RelA activity by dominant-negative IκBα, c-Raf, and MEKK1. CAT assays for analysis of κB reporter gene activities were performed as described previously (10) . The κB reporter gene plasmids were cotransfected into MDAPanc-28 and Capan-1 cells with various expression plasmids as indicated. The transfected cells were harvested, the relative transfection efficiencies were determined by using the cotransfected LacZ expression plasmid (1 μg, CMV-LacZ), and subsequent β-galactosidase activities in the cell extracts were very similar and used to normalize the transfection efficiencies. The results shown here are representative of five CAT assays. β-gl, β-galactosidase; WT, wild type. C, the β-actin promoter/β-galactosidase reporter gene plasmid was cotransfected into MDAPanc-28 and Capan-1 cells with various expression plasmids as indicated, and the relative transfection efficiencies were determined by using the cotransfected luciferase expression plasmid (1 μg, TK-Renilla luciferase), and subsequent β-galactosidase (β-gal) and Renilla luciferase (R. luc.) activities in the cell extracts were determined. Renilla luciferase activities were used to normalize the transfection efficiencies. Bars, SD. In D, inhibition of RelA activity by curcumin potentiates apoptotic cell death induced by Taxol. Twenty thousand cells/well were seeded in 96-well plates. After 8 h of incubation, cells in triplicate were either left untreated or treated with curcumin (50 μm), Taxol (10 μm), or both. At various time points as indicated, surviving cells were quantified by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. Bars, SD.
Volume 5, Issue 1
