1. Volume 39, Issue 2, Pages 171-178 (August 2003)
Downregulation of cytochromes P450 in growth-stimulated rat hepatocytes: role of c- Myc induction and impaired C/EBP binding to DNA 
Marina Tinel, Alain Berson, Johny Elkahwaji, Thierry Cresteil, Philippe Beaune, Dominique Pessayre 
Journal of Hepatology 
Volume 39, Issue 2, Pages (August 2003)
DOI: /S (03)

2. Fig. 1
EGF increases c-myc mRNA in cultured hepatocytes and this effect is prevented by a c-myc antisense P(S)ODN. Rat hepatocytes were cultured for 72 h after cell attachment. Lipofectin, with or without a sense or antisense c-myc P(S)ODN (2 μM), was added after cell attachment, and at 24 and 48 h. EGF (64 ng/ml) was added at 24 and 48 h. Total RNA was prepared, and c-myc mRNA and GAPDH mRNA were assessed by Northern blot analysis. The c-myc mRNA/GAPDH mRNA ratio (mean±SEM for five cultures) was quantified by laser densitometry of autoradiographs and expressed as a percentage of the mean ratio in control cells. *Different from control culture, P<0.05.
Journal of Hepatology  , DOI: ( /S (03) )

3. Fig. 2
EGF increases Myc/Max-binding to a DNA probe and this effect is prevented by c-myc antisense oligomers. Lipofectin, with or without a sense or antisense c-myc P(S)ODN (2 μM) (A) or a sense or scrambled c-myc PMO (2 μM) (B), was added after cell attachment and at 24 and 48 h. EGF (64 ng/ml) was added at 24 and 48 h. After 72 h of culture, nuclear proteins were prepared, and their binding to a Myc/Max-binding DNA probe was assessed with an electrophoretic mobility shift assay. Lanes 1, control; lanes 2, EGF; lanes 3, control+sense P(S)ODN or scrambled PMO; lanes 4, EGF+sense P(S)ODN or scrambled PMO; lanes 5, control+antisense P(S)ODN or PMO; lanes 6, EGF+antisense P(S)ODN or PMO.
Journal of Hepatology  , DOI: ( /S (03) )

4. Fig. 3
EGF decreases total CYP and this effect is prevented by a c-myc antisense P(S)ODN. Rat hepatocytes were cultured for 72 h after cell attachment. Lipofectin, with or without a sense or antisense c-myc P(S)ODN (2 μM), was added after cell attachment, and at 24 and 48 h. EGF (64 ng/ml) was added at 24 and 48 h. Total CYP (P450) (mean±SEM for four cultures) was measured from the absorbance of its CO complex in dithionite-reduced microsomes. *Different from control culture, P<0.05.
Journal of Hepatology  , DOI: ( /S (03) )

5. Fig. 4
A c-myc antisense PMO also prevents the EGF-mediated decrease in CYP2C11 and CYP3A proteins. Lipofectin, with or without a scrambled PMO or an antisense c-myc PMO (2 μM), was added after cell attachment and again at 24 and 48 h. EGF (64 ng/ml) was added at 24 and 48 h. At 72 h, cells were scraped and microsomes were prepared. Western blots of microsomal proteins (5 μg) were revealed with an anti-CYP2C11 or anti-CYP3A antibody, and protein bands (means±SEM for four cultures) were expressed as a percentage of the mean value in control cultures. Different from control cultures: *P<0.05; **P<0.001; ***P<
Journal of Hepatology  , DOI: ( /S (03) )

6. Fig. 5
Prevention of the EGF-mediated increase in c-myc mRNA and decrease in total CYP by retinoic acid or DMSO. Rat hepatocytes were cultured for 72 h after cell attachment. Retinoic acid (RA) (10 μM) or DMSO (2%) were added after attachment and at 24 and 48 h. EGF (64 ng/ml) was added at 24 and 48 h. At 72 h, cells were scraped to prepare total RNAs or microsomes. (A) The c-myc mRNA (mean±SEM for four cultures) was assayed by Northern blot analysis and expressed as a percentage of the mean value in control cultures. (B) Total CYP (P450) (mean±SEM for five cultures) was measured from the absorption of its CO complex in dithionite-reduced microsomes. *Different from control cultures, P<0.05.
Journal of Hepatology  , DOI: ( /S (03) )

7. Fig. 6
EGF does not change the expression of C/EBPα but increases C/EBPβ in a c-Myc-independent manner. Lipofectin, with or without a sense or antisense c-myc P(S)ODN (2 μM) was added after cell attachment and at 24 and 48 h. EGF (64 ng/ml) was added at 24 and 48 h. At 72 h, nuclear proteins were prepared. Western blots were revealed with a polyclonal anti-C/EBPα or anti-CEBPβ antibody, and C/EBP protein bands (mean±SEM for three cultures) were expressed as a percentage of the mean value in control cultures. *Different from control cultures, P<0.05.
Journal of Hepatology  , DOI: ( /S (03) )

8. Fig. 7
Co-immunoprecipitation of C/EBPα and C/EBPβ by an anti-c-Myc monoclonal antibody. Hepatocytes were cultured for 72 h after cell attachment, and treated or not with EGF (64 ng/ml) at 24 and 48 h. Nuclear proteins or buffer (blank) were immunoprecipitated with a monoclonal anti-c-Myc antibody. Eluted proteins were separated by SDS–polyacrylamide (10%) gel electrophoresis, transferred to nitrocellulose sheets, and revealed with a polyclonal anti-c-Myc, anti-C/EBPα or anti-C/EBPβ antibody (Ab). Note that the three proteins were present in the immunoprecipitate. Also note that the anti-C/EBP antibodies also recognize c-Myc.
Journal of Hepatology  , DOI: ( /S (03) )

9. Fig. 8
Effects of EGF and a c-myc antisense P(S)ODN or PMO on the binding of C/EBPs to a C/EBP-binding DNA probe. Lipofectin, with or without a sense or antisense c-myc P(S)ODN (2 μM) (A), or a scrambled or anti-sense c-myc PMO (2 μM) (B), was added after cell attachment and at 24 and 48 h. EGF (64 ng/ml) was added at 24 and 48 h. At 72 h, nuclear proteins were prepared, and the binding of C/EBPs to a C/EBP-binding DNA probe was assessed with an electrophoretic mobility shift assay. Lanes 1, control cultures; lanes 2, EGF; lanes 3, control+sense P(S)ODN or scrambled PMO; lanes 4, EGF+sense P(S)ODN or scrambled PMO; lanes 5, control+anti-sense P(S)ODN or PMO; lanes 6, EGF+anti-sense P(S)ODN or PMO. Note that EGF markedly decreased the binding of C/EBP dimers to the DNA probe (lanes 2 vs. lanes 1). This inhibitory effect was prevented in EGF-treated cells also treated with a c-myc antisense P(S)ODN or PMO (lanes 6).
Journal of Hepatology  , DOI: ( /S (03) )

10. Fig. 9
The N-terminal c-Myc domain (amino acids 1–262) prevents the binding of C/EBPs to a C/EBP-binding sequence on DNA. (A) Nuclear proteins (7 μg) were prepared from control hepatocytes and incubated with 0.05–0.25 μg of the N-terminal c-Myc domain. The binding of C/EBPs to a C/EBP-binding DNA probe was assessed by an electrophoretic mobility shift assay. Lane 1, control; lanes 2–5, 0.05, 0.06, and 0.25 μg of N-terminal c-Myc, respectively. (B) Control nuclear extracts were preincubated with 0.25 μg of the N-terminal c-Myc domain with or without a polyclonal, or a monoclonal anti-c-Myc antibody (4 μg). Lane 1, control; lane 2, c-Myc; lane 3, c-Myc+polyclonal antibody; lane 4, c-Myc+monoclonal antibody. The polyclonal, but not the monoclonal antibody, prevents the interaction of c-Myc with C/EBPs.
Journal of Hepatology  , DOI: ( /S (03) )