1. Volume 41, Issue 4, Pages 538-544 (October 2004)
Up-regulation of the high-affinity pyrimidine-preferring nucleoside transporter concentrative nucleoside transporter 1 by tumor necrosis factor-alpha and interleukin-6 in liver parenchymal cells 
Sonia Fernández-Veledo, Raquel Valdés, Ville Wallenius, F.Javier Casado, Marçal Pastor-Anglada 
Journal of Hepatology 
Volume 41, Issue 4, Pages (October 2004)
DOI: /j.jhep

2. Fig. 1
CNT1 protein expression in liver parenchymal and hepatoma cells. CNT1 protein expression was analyzed in crude extracts from adult rat hepatocytes (H), FAO cells (FAO), fetal rat hepatocytes (FH) and C2 cells (C2) by Western blot. Equal amounts of protein were loaded (20 μg) to allow direct comparisons of CNT1 levels among liver parenchymal cell models. A representative Western blot is shown.
Journal of Hepatology  , DOI: ( /j.jhep )

3. Fig. 2
Regulation of CNT1 protein in rat liver parenchymal cell models by the multifunctional cytokines, TNF-alpha and IL-6. Cells were treated with 10 ng/ml TNF-α and 20 ng/ml IL-6. CNT1 protein amounts were monitored by Western blot at the indicated times after the addition of the cytokine. Representative Western blots are shown. (A) Regulation of CNT1 in adult rat hepatocytes. Primary cultures were treated with the corresponding cytokines 24 h after seeding. Ten micrograms of protein were loaded for every Western blot. (B) Effect of cytokines in FAO, fetal hepatocytes (FH) and C2 cells. Cell cultures, at 70–75% confluence, were treated with either TNF-α or IL-6 and CNT1 protein amounts were again monitored at different time points (2, 5, 8, 12 and 24 h) by Western blot analysis. In this particular case, 20 μg protein were loaded for FAO cells, whereas 30 μg were used for fetal hepatocytes and C2 protein extracts. For simplicity, a representative immunoblot of protein extracts obtained 8 h after cytokine addition is shown. In the lower Figure in this panel, a representative experiment showing the effect of a mixture of 100nM Dexamethasone/1 μM thyroid hormone on CNT1 and CNT2 protein amounts in fetal hepatocytes is shown. The magnitude of the induction triggered by treatments was quantified densitometrically. Results (mean±SEM) are shown as arbitrary units normalized to control values (non-treated cells). Statistical significance was assessed using the Student's t test. Panel A: **,o,oP<0.01 for TNF-α and IL-6, respectively; Panel B: *P<0.05, ***P<0.001.
Journal of Hepatology  , DOI: ( /j.jhep )

4. Fig. 3
Effect of the multifunctional cytokines, TNF-α and IL-6, on CNT1 protein expression and related activity in primary cultures of rat hepatocytes. Twenty-four hours after cell seeding, monolayers were cultured, either in the absence or in the presence of 10 ng/ml TNF-α and 20 ng/ml IL-6, for a further 8 h. (A) Na+-dependent nucleoside uptake was measured using 1 μM cytidine as substrate and 1 min incubation time. Data are the mean±SEM of quadruplicate measurements made in three independent cultures. (B) Immunofluorescence of CNT1 protein in primary cultures of rat hepatocytes. Cells were treated with either TNF-α or IL-6, as detailed above. A representative immunofluorescence of non-treated hepatocytes (CTRL) is also shown.
Journal of Hepatology  , DOI: ( /j.jhep )

5. Fig. 4
Effect of TNF-α and IL-6 on the amount of CNT1 protein in liver parenchymal cell membranes. (A) A representative anti-CNT1 immunoblot using plasma (PM) and microsomal (MF) membrane fractions is shown. Cells were incubated either in the absence or in the presence of 10 ng/ml TNF-α and 20 ng/ml IL-6 for 8 h, and membrane fractions were prepared as described in Section 2. (B) Plasma and microsomal membrane fractions of hepatocytes were also checked for the enrichment in α1 subunit of the Na,K-ATPase pump and protein disulfide isomerase (PDI) primarily located in the ER lumen. As expected, plasma membranes are highly enriched in Na,K-ATPase, whereas, CNT1, which is known to be mostly located in subcellular vesicles, and PDI shows a significant enrichment in microsomal membrane fractions in control cells.
Journal of Hepatology  , DOI: ( /j.jhep )

6. Fig. 5
Effect of IL-6 on ERK 1/2 phosphorylation in adult rat hepatocytes. The activation of the extracellular regulated kinase (ERK) pathway by IL-6 was monitored in primary cultures of adult rat hepatocytes. Cell protein extracts were prepared, at different time points after cytokine addition, and monitored by Western blot for changes in the amounts of phosphorylated ERK 1/2 (upper panel). Total ERK 1/2 was also monitored (lower panel) as a control. Representative immunoblots and densitometric analysis of the gels (means±SEM) are shown.
Journal of Hepatology  , DOI: ( /j.jhep )

7. Fig. 6
Effect of PD98059 and wortmannin on the regulation of CNT1 by TNF-α and IL-6 in adult rat hepatocytes. (A) Cells were pre-incubated for 30 min with the ERK 1/2 inhibitor PD98059 (10 μM). Then, cells were incubated for 8 h either in the absence or in the presence of 10 ng/ml TNF-α and 20 ng/ml IL-6. A representative Western blot is shown. (B) Cells were pre-incubated with the IP3 kinase/Akt inhibitor wortmannin (100nM) and then treated with cytokines and processed as indicated above. Two representative Western blots are shown. The upper panel shows the effect triggered by TNF-α, whereas the lower one shows the IL-6 effects.
Journal of Hepatology  , DOI: ( /j.jhep )

8. Fig. 7
Regulation of CNT1 protein expression in TNF-α receptor-I and IL-6 knock-out mice. Mouse livers were homogenized as described in Section 2 and CNT1 protein amounts were monitored by Western blot analysis. For each condition, a representative immunoblot is shown (left Figures in each panel). Each lane corresponds to a different animal. (A) CNT1 protein amounts in liver extracts from IL-6 knock-out and wild-type mice. (B) CNT1 protein in livers from TNF-α receptor-I knock-out mice and effect of IL-6 treatment. Livers were sampled 24 h after a single injection of human recombinant IL-6 at a dose of 0.8 μg/g body weight. (C) Effect of IL-6 treatment on CNT1 protein amounts in livers from wild-type animals. Mice were treated with increasing concentrations of human recombinant IL-6 (range 40 ng to 1 μg) during a 21-day period. Control animals received injection of the vehicle [0.01M PBS and 0.1% BSA (FRACTION V; <0.1 ng endotoxin/mg; Sigma)]. The fold induction of each condition was calculated by densitometric analysis of the gels (means±SEM). Statistical significance using the Student's t test: *P<0.05; **P<0.01; ***P<0.001 (right-hand figures in the three panels).
Journal of Hepatology  , DOI: ( /j.jhep )

9. Fig. 8
Hypothetical model for the regulation of CNT1 expression by TNF-α and IL-6.
Journal of Hepatology  , DOI: ( /j.jhep )