DEV02820 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Inhibition of p53 or mTOR does not perturb cell cycle progression of E12.5 embryonic hepatocytes in vivo. The p53 inhibitor pifithrin-α (PFT) was injected into pregnant females at E10.5 and E11.5, hepatocytes were prepared from a pool of five to eight injected (or not) E12.5 wild-type embryos. Cell cycle progression was analyzed by FACscan of α-fetoprotein-positive cells. The top two panels show cell cycle distribution in a representative experiment. The table at the bottom reports the mean values±s.e.m. of the percentages of hepatocytes at the G0/G1, S, G2/M and subG0 phases from four independent experiments. To analyze the effect of rapamycin on cell cycle progression of E12.5 embryonic hepatocytes, rapamycin was injected into pregnant females as above. No significant difference in cell cycle progression was found in hepatocytes of rapamycin-treated mice compared to controls.

• Supplemental Figure 2 -

  Fig. S2. Hgf-induced Mdm2 translation, rather than protein stability, is cell-type specific and requires signals distinct from MAPKs and Src. (A) In contrast to embryonic hepatocytes, Hgf does not increase Mdm2 protein level in cortical neurons. (B) Rate of decay of Mdm2 protein in embryonic hepatocytes with basal Mdm2 levels (non-stimulated cells) as determined by cycloheximide treatments. Hgf treatment does not change Mdm2 protein stability. Mdm2 protein levels were quantified after normalization with tubulin, as cycloheximide treatment does not affect its half-life. (C) Inhibition of either MAPK or Src family members does not affect Mdm2 protein levels. Primary embryonic hepatocytes were stimulated with Hgf in the presence or absence of specific inhibitors for MEK/ERKs (PD 98059: 10 μM), p38 (SB 203580: 10 μM), JNK (L-JNKI 1: 10 μM) or Src family (SU 6656: 10 μM). Total extracts were blotted and probed with α-Mdm2 (top) and α-tubulin (bottom).

• Supplemental Figure 3 -

  Fig. S3. TNFα plus ActD treatment increases p53 protein expression and phosphorylation on S₁₈. (A) TNFα plus ActD treatment upregulates p53 protein levels. By contrast, Met does not alter p53 expression. After 24 hours of treatment, total extracts from embryonic hepatocyte cultures were blotted and probed with α-p53 (top), α-Mdm2 (middle), and re-probed with α-tubulin (bottom). Due to post-translational modification, p53 appears as a doublet in embryonic hepatocytes treated with TNFα plus ActD. Notice that the increased level of p53 is associated with the downregulation of Mdm2 expression compared with untreated cells. (B) TNFα plus ActD treatment induces p53 phosphorylation on amino-acid residue S₁₈ in a time-course dependent manner, and reduces Mdm2 protein levels. Total protein extracts were blotted and probed with α-Mdm2 (top) and α-phospho-S₁₈-p53 (bottom).