Figure 6
H19 inhibition increases nuclear localization of FoxO1 and impairs insulin signalling. (a) HepG2 cells were reverse transfected either with H19 siRNA (5 nM) or with the scramble (SCR), and after 48 h of incubation nuclear and cytoplasmic protein fractions were isolated as described in the “Methods” section. 30 µg protein from cytoplasmic and nuclear fraction was subjected to Western blot analysis using anti-FoxO1 antibody. GAPDH and Histone H3 were used as normalization controls for cytoplasmic and nuclear fractions, respectively. A representative blot is shown. Densitometric analysis of FoxO1 levels in the nuclear fractions of scramble and H19 siRNA transfected cells is also shown. Full length blots are presented in Supplementary Information (Supplementary Fig. S3). (b) HepG2 cells were plated on cover slips and reverse transfected either with H19 siRNA (5 nM) or the scramble (SCR) as described in “a”. After 48 h, cells were fixed and stained as described in the methods section with anti-FoxO1 antibody followed by Alexa Fluor-488 conjugated goat anti-rabbit IgG. Nuclei were counterstained with DAPI. Cells were visualized and levels of FoxO1 were quantified. Scale bar 10 µm. A representative figure is shown on the top and given below is the quantification of the nuclear levels of FoxO1. (c) HepG2 cells were transfected as in “a” and then incubated in the absence or presence of insulin (100 nM, 20 min). Cells were lysed, and the levels of total IR, pIR, total Akt and pAkt were estimated by Western blot analyses. β-Actin was taken as the normalization control. Full length blots are presented in Supplementary Information (Supplementary Fig. S4). Each value is the mean of three such experiments. Values presented are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. a.u., arbitrary units.
