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suppmat_1285_fig1.tif60KSerum withdrawal induces differential caspase-3 activation in wild-type and IRS-2-/- hepatocytes.<br /> (Upper panel)After incubation of IRS-2+/+ and IRS-2-/- neonatal hepatocytes in serum-free medium for several time periods, caspase-3 enzymatic activity was measured. Results are expressed as arbitrary units/mg of total protein and are means SE from three independent experiments with duplicate dishes. Statistical significance was carried out by Studentttest by comparison of IRS-2-deficient cells with wild-type cells (* P< .01).<br /> (Lower panel)Cells were cultured as described above. At the end of the culture time, cells were collected and cell lysates were analyzed by Western blot with the anti-active caspase-3 and anti-β actin antibodies. Representative autoradiograms are shown.
suppmat_1285_fig2.tif94KReconstitution with IRS-2 restores the survival effect of insulin in IRS-2-deficient neonatal hepatocytes.<br />A. 24 hours after infection of IRS-2-/- hepatocytes with adenoviruses encoding wild-type IRS-2 and β-gal, cells were cultured for a further 24 hours in serum-free medium with or without 100 nM insulin. The expression of Foxo1 was analyzed in nuclear extracts and the expression of Bcl-xL and Bim was analyzed in total cell extracts.<br />B. IRS-2-/- hepatocytes were infected and further cultured in serum free medium for 8 hours with or without 100 nM insulin. Then, caspase-3 activity was analyzed. Results are means SE from three experiments. Statistical significance was carried out by Studentttest by comparison of cells treated with insulin with the non-treated (* P< .01).<br />C. Cells were cultured as described in A. Extranuclear DNA was electrophoresed and visualized by UV fluorescence. Representative experiments are shown.

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