Fig 1: miR-150-IGF1R/IRS1 axis is a downstream target of FoxP3.A Venn diagram depicting the number of predicted transcription factors (blue) in 2-kb region upstream of miR-150 using the JASPAR database (relative score threshold >0.9) and the number of protein-coding genes positively correlated with miR-150 (orange) (r > 0.25 or >0.5, P < 0.05) in 265 OC tissues from TCGA. B Correlation between FoxP3 and mir-150 expression in 265 OC tissues from TCGA. C Diagram of the 2-kb region upstream of mir-150 promoter. The predicted FoxP3-binding sites (named A, B, C, D and E), and the DNA fragments incorporated into luciferase reporter constructs (named P1–P4) were indicated. Luciferase reporter assays with the corresponding reporter constructs in HEK293T cells were shown. D The relative levels of miR-150-5p and miR-150-3p in Lv-FoxP3-OC cells were determined by real-time RT-PCR. E, F Relative mRNA (E) and protein (F) levels of IRS1 and IGF1R in Lv-FoxP3-OC cells were measured by real-time RT-PCR and western blotting. G Correlation between FoxP3 and IRS1 or IGF1R expression in 265 OC tissues from TCGA. The data are presented as the mean ± s.d. *P < 0.05, **P < 0.01, ***P < 0.001 by Student’s t-test.
Fig 2: IRS1 and IGF1R are directly targets of miR-150-5p/3p.A Venn diagram depicting the number of the proteins significantly downregulated (blue) (FC < 0.833, P < 0.05) in Lv-mir-150-A2780 cells shown in iTRAQ analysis and the number of protein-coding genes negatively correlated with mir-150 (orange) (r < -0.25, P < 0.05) in 265 OC tissues from TCGA. B, C Luciferase activities were measured in HEK293T cells transfected with reporter plasmids containing the predicted binding sites, together with miR-150-5p (B), miR-150-3p (C) or control mimic. D, E Relative mRNA (D) and protein (E) levels of IRS1 and IGF1R in Lv-mir-150-OC cells were measured by real-time RT-PCR and western blot analyses. F The protein levels of IRS1 and IGF1R in SKOV3 and ES2 cells transfected with miR-150-5p or miR-150-3p mimic. G IHC staining showed that the expression of IRS1 and IGF1R was reduced in xenografted tumors from Lv-mir-150-ES2 cells. Magnification: ×400. Scale bar: 50 µm. The data are presented as the mean ± s.d. *P < 0.05, **P < 0.01, ***P < 0.001 by Student’s t-test.
Fig 3: XQ‐2d‐His‐SH2 CM‐(Arg)9 influenced multiple signaling cascades of PANC‐1 cells. (A) Inhibition of EGFR, VEGFR2, IGF‐1R, Src, AKT, and ERK1/2 phosphorylation by XQ‐2d‐His‐SH2 CM‐(Arg)9 at different incubation time or concentrations was examined in PANC‐1 cells. (B‐D) The panels showed reciprocal immunoprecipitation of EGFR and GRB2, IGF‐1R and IRS1, VEGFR2, and SHC in PANC‐1 cells treated as indicated above. IP, immunoprecipitation; IB, immunobloting. Data shown are representative of three independent experiments
Fig 4: The PI3K/AKT/mTOR pathway regulates the expression of FoxP3 and miR-150-5p/3p.A, B OC cells were treated with PI3K inhibitor (LY294002) or mTOR inhibitor (rapamycin) for 24 h. The mRNA (A) and protein (B) levels of FoxP3 were measured by real-time RT-PCR and western blot analyses, respectively. C The protein levels of IRS1, phosphorylated AKT (Ser473), total AKT, phosphorylated mTOR (Ser2448), total mTOR, and FoxP3 in Lv-IRS1-ES2 cells were determined by western blotting. D The protein expression levels of IRS1, FoxP3, phosphorylated mTOR (Ser2448), and total mTOR were measured in Lv-IRS1-ES2 cells treated with 10 nM rapamycin for 24 h. E, F The relative levels of miR-150-5p and miR-150-3p in SKOV3 and ES2 cells treated with LY294002 (E) or rapamycin (F). The data are presented as the mean ± s.d. *P < 0.05, **P < 0.01, ***P < 0.001 by Student’s t-test. G Schematic diagram of a feedback loop formed from the FoxP3-miR-150-IGF1R/IRS1 axis and PI3K/AKT/mTOR signaling pathway, and its function in OC tumorigenesis.
Fig 5: miR-150-IGF1R/IRS1 regulates PI3K/AKT/mTOR signaling in OC cells.A–D ES2 cells stably co-expressing miR-150, IGF1R and IRS1 were established. A The protein levels of IGF1R and IRS1 determined by western blotting. Forced IGF1R and IRS1 expression restored the effects of miR-150 on cell proliferation (B), cell apoptosis (C), and cell migration (D) in ES2 cells. Scale bar: 200 µm. E Functional annotation clustering of proteins dysregulated by miR-150 in A2780 cells was shown. The ten most enriched groups according to GO molecular function analysis are ranked based on combined score. F The protein levels of IGF1R, IRS1, phosphorylated IRS1 (Ser307), phosphorylated AKT (Ser473), total AKT, phosphorylated mTOR (Ser2448), and total mTOR in ES2 cells transfected with siIGF1R or siIRS1 were determined by western blotting. G Effects of overexpression of miR-150 on protein levels of IGF1R, IRS1, phosphorylated AKT (Ser473), total AKT, phosphorylated mTOR (Ser2448), and total mTOR in OC cells. H Forced IGF1R and IRS1 expression partially restored the levels of phosphorylated AKT (Ser473) and phosphorylated mTOR (Ser2448) reduced by miR-150. The data are presented as the mean ± s.d. **P < 0.01, ***P < 0.001, by Student’s t-test.
Supplier Page from Proteintech Group Inc for IGF1R antibody