1. Volume 136, Issue 5, Pages 1659-1668 (May 2009)
Interaction of Cyclooxygenase-2 Variants and Smoking in Pancreatic Cancer: A Possible Role of Nucleophosmin 
Dan Zhao, Dongkui Xu, Xuemei Zhang, Li Wang, Wen Tan, Yongli Guo, Dianke Yu, Hui Li, Ping Zhao, Dongxin Lin 
Gastroenterology 
Volume 136, Issue 5, Pages (May 2009)
DOI: /j.gastro
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2. Figure 1
Promoter activities between constructs with different alleles in the absence or presence of cigarette smoke condensate (CSC) stimulation. Two COX-2 promoter constructs containing −765G or −765C were transiently transfected to HCT-116, PANC-1, and AsPC-1 cells. After transfection for 12 hours, CSC was added at 2.0 μg/mL in fresh serum-free medium and incubated for an additional 12 hours. The promoter activities were measured by dual luciferase assays, and the results were expressed as relative luciferase activity (RLA) of the COX-2 reporter constructs compared with the empty pGL3-Basic vector. Three independent transfection experiments were performed, and each was done in triplicate. *Constructs with the C allele had higher activities compared with those with the G allele (P < .001).
Gastroenterology  , DOI: ( /j.gastro )
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3. Figure 2
Electrophoretic mobility shift assays using biotin-labeled probes and nuclear extracts from HeLa and PANC-1 cells. (A) HeLa nuclear extracts were incubated with biotin-labeled G or C allele. The G allele showed 1 DNA-protein complex (Band I), but the C allele showed 2 DNA-protein complexes (Band I and Band II). The competition assays by the addition of 5-, 25-, or 125-fold molar excess of unlabeled G probe to the incubation mixture showed that Band I but not Band II could be inhibited. (B) PANC-1 nuclear extracts were incubated with biotin-labeled G or C allele. The G allele showed no DNA-protein complex, but the C allele showed 1 DNA-protein complex (Band II), which could not be inhibited by the addition of unlabeled G probe. (C) Competition assays with both unlabeled G or C probe using HeLa nuclear extracts. Band I was eliminated by the addition of excessive unlabeled G probe, and Bands I and II were eliminated by the addition of excessive unlabeled C probe, suggesting that Band II is C allele-specific. (D) Supershift assays with biotin-labeled C allele and HeLa nuclear extracts in the presence of NPM or p-NPM antibody. NPM antibody depleted Band II formation (Lane 3), but the supershifted band (SS) was detected only when p-NPM antibody was used (Lane 4).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2009 AGA Institute Terms and Conditions

4. Figure 3
CSC stimulation caused decrease in nuclear p-NPM and p-NPM bound to the COX-2 promoter. (A) Chromatin immunoprecipitation assays using antibodies against NPM, p-NPM, NF-κB p65, and actin. The presence of the COX-2 promoter was examined by PCR. Lane 1, with CSC treatment; Lane 2, without CSC treatment; M, DNA molecular marker. (B) Immunofluorescent analysis of p-NPM (green) and COX-2 (red) in HCT-116 cells treated without CSC (upper panel) or with CSC (lower panel). Nucleuses were visualized by DAPI (blue). (C) The impact of CSC treatment on NPM, p-NPM, and COX-2 expression and distribution in nucleus and cytoplasm. Cells were treated with or without CSC, and nuclear and cytoplasmic proteins were isolated. NPM, p-NPM, and COX-2 levels were determined by Western blot. Decreased ratio of nuclear NPM or p-NPM level to cytoplasm NPM or p-NPM level was associated with increased COX-2 expression after CSC treatment. Nu, nuclear; Cy, cytoplasmic. (D) Knockdown of NPM by RNAi caused decrease in COX-2 expression. Cells were transfected with RNA duplexes homology to NPM RNA, NPM-siRNA1, or NPM-siRNA2, or a control RNA duplex, and the proteins in cell lysates were analyzed by Western blot.
Gastroenterology  , DOI: ( /j.gastro )
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