Fig 2: Hepatic ablation of Ide alters multiple intracellular insulin-signaling pathways. (A) Representative western blots depicting the effects of insulin on total or phosphorylated IDE, IR, AKT1, AKT2, GSK3a, and GS. Expression of GAPDH was determined to ensure similar protein levels for total liver lysates. Two representative samples have been used for the final arrangement of the figure. Densitometric analyses of the data in panel A. The y-axis represents the ratio of total protein or phosphorylated versus GAPDH, and phosphorylated versus total protein in arbitrary units (AU) for IR (B), p-IR (C), p-IR/IR (D), AKT1 (E), p-AKT1Ser473 (F), p-AKT1Thr308 (G), p-AKT1Ser4731/AKT1 (H), p-AKT1Thr308/AKT1 (I), AKT2 (J), p-AKT2Ser474 (K), p-AKT2Ser474/AKT2 (L), GSK3a (M), p-GSK3a (N), p-GSK3a/GSK3a (O), GS (P), p-GS (Q), p-GS/GS (R) in WT and L-IDE-KO mice. Data are mean ± SEM. n = 5 per genotype and condition. *p value <0.05 vs. saline; $p value <0.05 vs. WT by ANOVA. (S) Representative immunoblot using anti-GLUT2 antibodies. Densitometric quantification of GLUT2 (T). Data are mean ± SEM. n = 4 per group. *p value <0.05 vs. WT.

Fig 3: Effect of cixutumumab (A12) on the IGF1-stimulated INSR, IGF1R, AKT, and ERK phosphorylations in C4-2 cells. (A and B) Effect of A12 on the IGF1-induced INSR and IGF1R phosphorylations. C4-2 cells were treated with A12 for 24 h, in the presence of IGF1 (50 or 100 ng/ml) during the last 10 min of incubation. At the end of the incubation period, the cells were lysed and immunoprecipitated with INSR (A) or IGF1R (B) antibodies, electrophoresed through SDS–PAGE, and immunoblotted with anti-phospho-IGF1R/INSR, anti-total INSR, or anti-total IGF1R. (C and D) Scanning densitometric analysis of phospho-INSR and phospho-IGF1R. Optical density is expressed as phospho-INSR (C) or phospho-IGF1R (D) values normalized to the corresponding total INSR or IGF1R levels. Bars represent means±s.e.m. of three independent experiments. *P<0.05 vs untreated cells (C) and *P<0.05 vs A12+IGF1-treated cells (D). (E and F) Effect of A12 on the IGF1-induced AKT and ERK1/2 phosphorylations. C4-2 cells were processed as described above, lysed, electrophoresed, and immunoblotted with anti-phospho-AKT (E) or phospho-ERK1/2 (F) and total AKT or total ERK1/2. (G and H) Scanning densitometric analysis of phospho-AKT and phospho-ERK1/2. Optical density is expressed as phospho-AKT (G) or phospho-ERK1/2 (H) values normalized to the corresponding total AKT or ERK1/2 levels. Bars represent means±s.e.m. of three independent experiments. *P<0.05 vs control cells (G) and *P<0.05 vs A12+IGF1-treated cells (H). Results of typical experiments, repeated three times with similar results, are shown in the figure.

Fig 4: Drug screening identifies ceritinib and dasatinib as potent inhibitors of OS proliferation in vitro. (a) Primary tumor cells of six patients (corresponding to seven samples) and the HOS cells were incubated with the indicated inhibitors. After nine days, the cell proliferation was measured. The ratio between the absorbance at day nine and the absorbance at day zero is indicated. At least two independent experiments were used to calculate the ratio and each experiment was done in triplicates. Low proliferation is outlined in red, middle in orange, and strong in blue. (b) The primary tumor cells of sample 386 were incubated with the indicated inhibitors. After nine days, the cell proliferation was measured and the ratio between the absorbance at day nine and the absorbance at day zero was calculated. The proliferation experiments were carried out in biological duplicates. Data are represented as the mean ± Standard deviation (SD). (c) 386 cells were stimulated after starvation with IGF2. The expression of IGF1R, INSR, AKT serine/threonine kinase 1 (AKT), extracellular-signal regulated kinases (ERK), kinase Src (Src) and their phosphorylated (P) form was analyzed by Western blot with specific antibodies. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as loading control. A representative experiment of three independent experiments is shown. Densitometric analysis of two independent experiments is shown. The control (unstimulated) condition was normalized to one.

Fig 5: Functional interactions between INSR/IGF1R and p53 in regulation of INSR promoter activity.(A) IGF1R-KD, INSR-KD and control MCF7 cells were co-transfected with the pGL3(-877/-2) LUC INSR promoter luciferase reporter along with an INSR-A expression vector (INSR-GFP) (or empty pEGFP vector) and a wild-type p53 expression vector (or empty pCMV). After 48 h cells were harvested and INSR promoter activity was measured as described in the legend to Figure 2. The left inset presents the basal INSR promoter activity levels at a large scale. Total, cytoplasmic and nuclear fractions of IGF1R-KD cells were immunoprecipitated with an INSR antibody, electrophoresed and immunoblotted with anti-INSR and anti-p53 (right inset). IgG was used as a control for the co-IP experiment. * p < 0.01 versus control cells. § p < 0.01 versus empty pCMV-transfected cells. (B) Cells were transfected with the INSR promoter reporter along with an IGF1R expression vector (or empty pEGFP) and a p53 expression vector (or empty pCMV). Cells were processed as described above. Total, cytoplasmic and nuclear fractions of INSR-KD cells were immunoprecipitated with an IGF1R antibody, electrophoresed and immunoblotted with anti-IGF1R and anti-p53 (inset).