Figure 5

Insulin-induced HMGA1/FoxO1 phosphorylation and their kinetics. (a) Time lapse imaging of the intranuclear distribution of CFP-FoxO1 in HEK-293 cells, either untransfected (control), or transfected with HMGA1 or HMGA1m, after treatment (0 and 20 min) with insulin. Pictures are optical sections made with a confocal laser scanning microscope. Bars correspond to 2 µm. Representative images of CFP-FoxO1 in HEK-293 cells relative to the FRAP experiment reported in (b). (b) FRAP analysis of FoxO1 in HEK-293 cells overexpressing HMGA1 wild-type or mutant HMGA1m. FRAP curves of CFP-FoxO1 in HEK-293 cells, either untreated (left) or treated with 10 nM insulin for 20 min (right). FRAP curves were calculated as described in Methods. (c) Time-course of the effect of insulin on FoxO1 phosphorylation in HEK-293 cells overexpressing HMGA1 (left) or HMGA1m (right). Representative WBs of HMGA1 and nuclear and cytosolic phosphorylated pFoxO1 are shown in both conditions. A comparison of the nuclear phosphorylated form of FoxO1 in cells overexpressing either HMGA1 (black bars) or HMGA1m (gray bars), under insulin treatment, is shown in bar graphs. Data are shown as the means ± s.e.m. of three separate experiments. Cropped blots are shown in the figure. Full-length WBs are presented in Supplementary Fig. S1. *P < 0.05 vs basal untreated cells (0 min). (d) Occupancy of the IGFBP1 gene promoter by FoxO1, as measured by ChIP in HEK-293 cells transfected with either HMGA1 or HMGA1m expression vectors, in the absence (−) or presence (+) of insulin. A representative assay is presented. qRT-PCR of ChIP-ed samples is shown in each condition. Cropped gel is shown in the figure. Full-length ChIP is presented in Supplementary Fig. S1. *P < 0.05 vs insulin untreated cells transfected with wild-type HMGA1 effector plasmid (slashed bar). Immunoblots for the expression of nuclear HMGA1 and pFoxO1 in each condition are shown in the autoradiograms. Cropped blots are shown in the figure. Full-length WBs are presented in Supplementary Fig. S1. (e) mRNA and protein expression levels of IGFBP1 and HMGA1 in HEK-293 cells transfected with either HMGA1 or HMGA1m expression vector in the absence (−) or presence (+) of insulin. Representative WBs are shown. Data are means ± s.e.m. of three separate experiments, each in triplicate. *P < 0.05 vs insulin untreated (HMGA1) cells. Cropped blots are shown in the figure. Full-length WBs are presented in Supplementary Fig. S1.