Fig 1: Effects of IGF-1-/IGF-2-neutralizing antibody on invasion and stemness of co-cultured cells. The co-culture system of C643 cells and M2-like TAMs was cultured in RPMI-1640 medium containing IGF-1-/IGF-2-neutralizing antibody (2 µg/ml) for 24 h. (A and B) The invasive ability of C643 cells was determined by a Transwell assay. (C and D) The cancer stemness of C643 cells was determined by a tumour sphere-formation assay. (E and F) Epithelial-mesenchymal transition and cancer stemness markers of C643 cells were examined by western blotting. The experiment was repeated three times, and the results are shown as the mean ± standard deviation. *P<0.05 and **P<0.01. TAM, tumour-associated macrophage; IGF, insulin-like growth factor.
Fig 2: Effects of IGF-1-/IGF-2-neutralizing antibody on IR-A/IGF-1R-mediated PI3K/AKT/mTOR signalling in co-cultured cells. The co-culture system of C643 cells and M2-like TAMs was cultured in RPMI-1640 medium containing IGF-1-/IGF-2-neutralizing antibody (2 µg/ml) for 24 h. (A and B) The expression of IR-A/IGF-1R/PI3K/AKT signalling pathway proteins in C643 cells was examined by western blotting. C643 cells were cultured alone as the control groups. The experiment was repeated three times, and the results are shown as the mean ± standard deviation. *P<0.05 and **P<0.01. TAM, tumour-associated macrophage; IGF, insulin-like growth factor; IR, insulin receptor; IGF-1R, IGF1 receptor; p-, phosphorylated.
Fig 3: Effects of exogenous IGF-1/IGF-2 on the invasion and stemness of C643 cells. (A and B) Invasive ability was determined using a Transwell assay in C643 cells treated with IGF-1 or IGF-2. (C and D) The cancer stemness of C643 cells was determined by a tumour sphere-formation assay. (E and F) epithelial-mesenchymal transition and cancer stemness markers of C643 cells were examined by western blotting. The experiment was repeated three times, and the results are shown as the mean ± standard deviation. *P<0.05 and **P<0.01 vs. control. TAM, tumour-associated macrophage; IGF, insulin-like growth factor.
Fig 4: Effects of M2-like TAM-secreted IGF-1 and IGF-2 on the activation of IR-A/IGF-1R signalling in anaplastic thyroid carcinoma cells. (A and B) The IGF-1 and IGF-2 mRNA levels in C643 cells, M2-like TAMs and co-cultured cells were measured by reverse transcription-quantitative PCR. (C and D) The concentrations of IGF-1 and IGF-2 in supernatants obtained from C643 cells, M2-like TAMs and co-cultured cells were measured by ELISA. (E and F) The protein expression levels of IR-A, IGF-1R, p-IR-A and p-IGF-1R in C643 cells were measured by western blotting after co-culture with M2-like TAMs for 24 h. C643 cells were cultured alone as the control groups. The experiment was repeated three times, and the results are shown as the mean ± standard deviation. *P<0.05 and **P<0.01. TAM, tumour-associated macrophage; IGF, insulin-like growth factor; IR, insulin receptor; IGF-1R, IGF1 receptor; p-, phosphorylated.
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