1. Volume 119, Issue 2, Pages 493-501 (August 2000)
Regulation of cyclooxygenase 2 expression in hepatocytes by CCAAT/enhancer-binding proteins 
Nuria A. Callejas, Lisardo Boscá, Christopher S. Williams, Raymond N. DuBois, Paloma Martín-Sanz 
Gastroenterology 
Volume 119, Issue 2, Pages (August 2000)
DOI: /gast
Copyright © 2000 American Gastroenterological Association Terms and Conditions

2. Fig. 1
Expression of COX-2 in prenatal and postnatal hepatocytes stimulated with LPS. Hepatocytes were obtained from livers of animals at the indicated gestation period and postnatal age and maintained in culture. Cells were stimulated for 24 hours with 1 μg/mL LPS, and the amount of COX-2 and NOS-2 was determined by Western blot analysis. Nonstimulated cells did not express NOS-2 and COX-2. C/EBP-α was determined in nuclear extracts obtained at 0 hour after LPS challenge. Results are the densitometric analysis of the band intensities corresponding to the indicated proteins from 4 experiments (mean ± SEM). *P < vs. samples at day 22 of gestation.
Gastroenterology  , DOI: ( /gast )
Copyright © 2000 American Gastroenterological Association Terms and Conditions

3. Fig. 2
Transfection of fetal and adult hepatocytes with sequences of the rat COX-2 promoter. Hepatocytes were transfected with a β-galactosidase–expression vector and the indicated plasmids encoding a luciferase reporter gene. Cells were stimulated for 24 hours with 1 μg/mL LPS. Luciferase activity was expressed with respect to the β-galactosidase activity for each condition. Results are the mean ± SEM of 4 experiments assayed per duplicate. *P < 0.01 vs. corresponding condition in the absence of LPS challenge.
Gastroenterology  , DOI: ( /gast )
Copyright © 2000 American Gastroenterological Association Terms and Conditions

4. Fig. 3
Recovery of COX-2 expression in adult hepatocytes maintained in culture for prolonged periods. Adult hepatocytes were maintained in culture for the indicated times. Cells were stimulated for 24 hours in the absence or presence of 1 μg/mL LPS at the indicated days. (A) The corresponding protein levels of COX-2, NOS-2 (at 24 hours), and C/EBP-α (at 0 hour) were determined. (B) RNA levels of COX-2 were determined at the indicated times in cultured cells stimulated with LPS. (C) Release of nitrite plus nitrate and PGE2 was determined in the culture medium after a 24-hour incubation with LPS (•, ■). 100% of NOx and PGE2 correspond to 22 nmol and 37 ng/mg protein of each, respectively. ○, 2, Cells nontreated with LPS. (D) After 7 days in culture, hepatocytes were analyzed by immunocytochemistry to determine the percentage of albumin-positive cells and COX-2–expressing cells after 24 hours of stimulation with LPS. Results are the mean ± SEM of 3 experiments. *P < vs. control condition (D).
Gastroenterology  , DOI: ( /gast )
Copyright © 2000 American Gastroenterological Association Terms and Conditions

5. Fig. 4
Transient expression of C/EBP-α impairs COX-2 induction in AT3F cells. Adult and fetal hepatocytes and AT3F cells were incubated for 24 hours with 1 μg/mL LPS. (A) The levels of COX-2 (at 24 hours) and C/EBP-α and C/EBP-δ (at 0 hours) were determined by Western blot analysis. (B) The synthesis of PGE2 was analyzed in the culture medium. (C) AT3F cells were cotransfected with a plasmid encoding the −446 to +32 sequence of the COX-2 promoter linked to a luciferase gene (see Figure 2), and the indicated expression vectors containing the C/EBPs or the void vector. After 24 hours of stimulation in the absence or presence of LPS, luciferase activity was measured and expressed. Results are the mean ± SEM of 3 experiments. *P < vs. cells transfected with the void pEMBL19 plasmid (C).
Gastroenterology  , DOI: ( /gast )
Copyright © 2000 American Gastroenterological Association Terms and Conditions

6. Fig. 5
Binding of nuclear proteins to CCAAT enhancer (NF-IL6 binding site) and κB motif of the rat COX-2 promoter. Nuclear extracts were prepared from control and LPS-treated cells (1 μg/mL, 1 hour). (A) The oligonucleotide sequences corresponding to the NF-IL6 and NF-κB binding sites of the rat COX-2 promoter were assayed by EMSA. Supershift experiments with anti-C/EBP antibodies were performed using nuclear extracts of fetal and adult hepatocytes and AT3F cells. (B) Results show a representative blot of 3.
Gastroenterology  , DOI: ( /gast )
Copyright © 2000 American Gastroenterological Association Terms and Conditions