Fig 1: Expressions of IGF-2 and its receptors in HemSCs and the effect of IGF-2 on the adipogenesis of HemSCs. (A) Immunohistochemistry of HemSCs using antibodies specific to IGF-2, IGF-2R, IGF-1R, and IR. (B) IGF-2 induced differentiation of HemSCs into adipocytes. HemSCs that were cultivated for 10 days in adipogenic differentiation media; adipogenic differentiation media with IGF-2 (100 ng/ml); adipogenic differentiation media with IGF-2 (100 ng/ml) plus OSI-906 (1 µM). After Oil Red O-staining, phase-contrast microscopy was used to observe lipid droplets. Scale bar, 100 µm. IGF-2, insulin-like growth factor-2; HemSC, hemangioma stem cell; IGF-1R, IGF-1 receptor; IR, insulin receptor.
Fig 2: Effects of IGF-2 on the expression of adipogenic markers in HemSCs. The C/EBPa, C/EBPß, PPAR, and adiponectin protein levels were analyzed by western blot analysis. (A) The results showing protein levels in untreated and treated HemSCs for C/EBPa, C/EBPß, PPAR?, and adiponectin in IGF-2 (100 ng/ml), IGF-2 (100 ng/ml) with OSI-906 (1 µM), IGF-2 (100 ng/ml) with LY294002 (10 µM), and LY294002 (10 µM). ß-actin was used as a loading control. (B) Western blot analysis showed p-AKT and total AKT as well as ß-actin protein bands in confluent HemSCs cultures exposed to the indicated treatments for 1 h. Groups: No treatment; IGF-2 (100 ng/ml); IGF-2 (100 ng/ml) plus OSI-906 (1 µM); IGF-2 (100 ng/ml) plus LY294002 (10 µM). Quantification of the p-AKT protein levels showed an obvious increase in the IGF-2 group (P<0.05). No changes were detected in the total AKT protein levels among the groups (P>0.05). Values are mean ± SD. n=3, *P<0.05 vs. Control group. #P<0.05 vs. IGF-2. IGF-2, insulin-like growth factor-2; HemSC, hemangioma stem cell; p-AKT, phosphorylated AKT.
Fig 3: Analysis of the adipogenic gene and protein expression of propranolol-treated HemSCs. (A) Western blot analysis results of protein levels in propranolol-treated HemSCs for C/EBPa, C/EBPß, and PPAR in 0, 50, and 100 µM. (B) Quantitative RT-PCR was performed to analyze the expression of C/EBPa, C/EBPß, and PPAR. (C) Western blot analysis showed IGF-2 and ß-actin protein bands in propranolol-treated HemSCs. Values are mean ± SD. n=3; **P<0.01 vs. Control group. HemSC, hemangioma stem cell; IGF-2, insulin-like growth factor-2.
Fig 4: IGF-2 stimulated the proliferation of HemSCs in vitro. (A) IGF-2 promoted the proliferation of HemSCs in vitro; IGF-2 at 100 and 200 ng/ml significantly promoted the proliferation of HemSCs as compared with the control group, and IGF-2 at 100 ng/ml exhibited the highest proliferation activity among all groups. (B) Growth curves of IGF-2 treated HemSCs. The proliferation of IGF-2-treated HemSCs (days 1–7) was promoted compared with the untreated group. Values are mean ± SD, n=3; *P<0.05 and **P<0.01 vs. Control group; #P<0.05 vs. 100 ng/ml. IGF-2, insulin-like growth factor-2; HemSC, hemangioma stem cell.
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