1. Volume 131, Issue 1, Pages 223-232 (July 2006)
S–Adenosylmethionine Regulates Cytoplasmic HuR Via AMP–Activated Kinase 
María L. Martínez–Chantar, Mercedes Vázquez–Chantada, Marta Garnacho, M. Ujue Latasa, Marta Varela–Rey, Javier Dotor, Monica Santamaria, Luis A. Martínez–Cruz, Luis A. Parada, Shelly C. Lu, José M. Mato 
Gastroenterology 
Volume 131, Issue 1, Pages (July 2006)
DOI: /j.gastro
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

2. Figure 1
SAM blocks the phosphorylation and activation of AMPK stimulated by HGF or AICAR. (A) Rat hepatocytes were incubated for 4 hours with HGF (25 ng/mL) or HGF + SAM (4 mmol/L) and (B) with AICAR (2 mmol/L) or AICAR + SAM (4 mmol/L). The cell extract (30 μg per lane) was collected and analyzed by Western blotting with the indicated antibodies. (C) AMPK activity in rat hepatocytes was determined using the SAMS peptide assay in the absence (open bars) or presence (solid bars) of SAM at the concentrations described in A. The data are expressed as fold increase over the control value. (D) Hepatocytes were preincubated for 30 minutes in the absence or presence of calyculin (5 nmol/L). HGF, AICAR, and SAM were added as mentioned in A. Western blotting procedure was performed, and the blot was subsequently incubated with an antibody against the phosphorylated form of AMPK (T172).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

3. Figure 2
AMPKα1 interacts with PP2A regulatory subunit A. Rat hepatocytes were incubated with AICAR (2 mmol/L), SAM (4 mmol/L) or AICAR + SAM, or AICAR + SAM + calyculin A (5 nmol/L) for 4 hours. Calyculin A was added 30 minutes before other additives. Total crude extract from hepatocytes was inmunoprecipitated with anti-PP2A regulatory subunit A and screened for the presence of AMPKα1 (upper panel). Supernatant and crude extracts in the input (30 μg) (middle and lower panels) were immunodetected with an antibody against AMPKα1 or PP2A. A volume of 2 μL anti-PP2A was used for the immunoprecipitation.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

4. Figure 3
Effect of AMPK activators and SAM on the subcellular localization of HuR. (A) Western blot analysis of HuR levels in cytoplasmic (40 μg), nuclear (20 μg), and whole cell (20 μg) lysates prepared from rat hepatocytes that were treated for 4 hours either with AICAR (2 mmol/L) and AICAR + SAM (4 mmol/L) (upper panel) or HGF (25 ng/mL) and HGF + SAM (4 mmol/L) (lower panel). β-Tubulin and HDAC1 were used as a loading control in the subcellular fractions. (B) Immunofluorescent detection of HuR in rat hepatocytes that were either left untreated or treated for 4 hours with the combination of AICAR, HGF, or SAM, mentioned in A (upper panel). Hoechst staining to visualize nuclei (lower panel).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

5. Figure 4
MLP-29 liver cells expressing reduced AMPKα1 show reduced cytoplasmic HuR in response to AICAR. MLP-29 cells were transfected with 0.6 μmol/L AMPKα1 siRNA or control siRNA using oligofectamine reagent. Twenty-four hours after transfection, cells were treated with AICAR (2 mmol/L) or AICAR + SAM (4 mmol/L). (A) Immunoblots of AMPKα1 protein expression and phosphorylated pT172AMPKα1 in MLP-29 cells transfected with (1) control siRNA or (2) AMPKα1 siRNA. (B) Immunofluorescent detection of HuR in MLP-29 cells transfected with control siRNA or AMPKα1 siRNA. Cells were either left untreated or treated for 4 hours with AICAR or the combination of AICAR + SAM.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

6. Figure 5
SAM blocks AICAR-induced HuR binding to cyclin A2 mRNA and HuR-mediated stabilization and expression of this messenger. (A) RT-PCR analysis of mRNA isolated from rat hepatocytes treated with AICAR (2 mmol/L) or AICAR + SAM (4 mmol/L) for 24 hours. The graph shows ethidium bromide-stained agarose gels (1%) containing PCR products of cyclin A2 mRNA amplified from 2 μg total RNA. A representative experiment carried out in triplicate is shown. (B) Western blot analysis to determine cyclin A protein expression in rat hepatocytes treated as indicated in A. Equal protein loading was assured by actin Western blot. A representative experiment carried out in triplicate is shown. (C) Cell lysates from AICAR (2 mmol/L), AICAR + SAM (4 mmol/L), or nontreated hepatocytes were inmunoprecipitated with HuR or IgG (control) antibodies. Bound RNA was harvested with the guanidinium thyocianate method 4 hours posttreatment, reverse transcriptased, and PCR amplified with cyclin A2 or cyclin D1 primers. Actin was used as a negative control of the specificity of the inmunoprecipitation (not shown). A representative experiment carried out in triplicate is shown. (D) After treatment with AICAR (2 mmol/L) for 4 hours, rat hepatocytes were washed and incubated with media containing actinomycin D (2 μg/mL) for 4.5 hours in the presence of AICAR (solid circles) or AICAR + SAM (open circles, 4 mmol/L). At the indicated time, cyclin A2, cyclin D1, and actin mRNA levels were determined by RT-PCR normalized to the internal control GADPH and plotted on a logarithmic scale. In the case of cyclin A2, the data point at 30 minutes in the presence of AICAR + SAM was shown to be an outlier (P < .05) and, accordingly, was not included in the regression analysis.22 The scientific statistical R program ( was used to perform the outlier test as described in Dalgaard.23
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

7. Figure 6
AICAR induces proliferation in isolated rat hepatocytes. Subconfluent hepatocytes were serum starved overnight and stimulated with buffer only, AICAR (2 mmol/L), AICAR + SAM (4 mmol/L), or SAM (4 mmol/L) for 24 hours. For the final 3 hours of stimulation, BrdU was added to the medium for labeling. Cells were fixed and stained for BrdU and then scored for BrdU-positive nuclei.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

8. Figure 7
AMPKα1 subunit phosphorylation and the subcellular localization of HuR are impaired in liver specimens from wild-type and MAT1A knockout mice. (A) Liver extracts (15 μg per lane) from 8-month-old wild-type and MAT1A knockout (MAT1A−/−) mice were analyzed by Western blotting with anti pT172AMPKα1 antibody. AMPKα1 is shown as a loading control. (B) Western blot analysis of HuR levels in cytoplasmic (40 μg), nuclear (20 μg), and whole liver (20 μg) extracts prepared from wild-type and MAT1A knockout (MAT1A−/−) mice. β-Tubulin and HDAC1 were used as loading controls in the subcellular fractions.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

9. Figure 8
Binding of HuR to HuR-target mRNA and the expression of these mRNAs are increased in MAT1A knockout mice. (A) Liver extracts (250 μg per lane) from 8-month-old wild-type and MAT1A knockout (MAT1A−/−) mice were immunoprecipitated with HuR or IgG (control) antibodies. Bound RNA was harvested with guanidinium thyocianate, reverse transcriptased, and PCR amplified with cyclin A2, cyclin D1, or cyclin E primers. PCR products were visualized by electrophoresis in ethidium bromide-stained agarose gels. (B) The abundance of the transcripts present in liver extracts after HuR immunoprecipitation was assed, and fold differences were plotted. Input, total mRNA in liver extract; unbound, unbound mRNA after immunoprecipitation with HuR antibody; bound, bound mRNA after immunoprecipitation with HuR antibody; control, bound mRNA after immunoprecipitation with IgG antibody.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions