Fig. 6: IGF-1-IGF-1R signaling upregulates FOXC1 expression via PI3K/AKT/NF-κB; ERK/NF-κB pathway.

A, B KYSE-150 cells were treated with IGF-1(50 ng/mL) for 12 h in the presence or absence of 10 μM、20 μM IGF-1R inhibitor Linsitinib, the protein expression of FOXC1, phosphorylated and total IGF-1R was detected by western blot analysis (A) and the mRNA expression of FOXC1 was detected by qPCR analysis (B). C-E.KYSE-150 cells were transiently transfected with control or p65 siRNA for 48 h and then treated with IGF-1(50 ng/mL) for 12 h. The protein level of p65, FOXC1 was detected by western blot analysis (C) and the mRNA of p65(D),FOXC1 (E) was detected by qPCR analysis. F, G KYSE-150 cells were treated with IGF-1(50 ng/mL) for 12 h in the presence or absence of 10 μM、20 μM BAY11-7082, the protein expression of FOXC1, phosphorylated and total p65 was detected by western blot analysis (F) and the mRNA expression of FOXC1 was detected by qPCR analysis (G). H The ChIP assay showed the direct binding abilities of p65 to the FOXC1 promoter induced by IGF-1, and p65 inhibitor BAY11-7082 reduced the binding abilities of p65 to the FOXC1 promoter. I, J KYSE-150 cells were treated with IGF-1(50 ng/mL) for 12 h in the presence or absence of 10 μM GDC-0991,10 μM UO126, the protein expression of FOXC1,phosphorylated and total p65, phosphorylated and total Akt and ERK1/2 was detected by western blot analysis (I) and the mRNA expression of FOXC1 was detected by qPCR analysis (J). K The ChIP assay showed the direct binding abilities of p65 to the FOXC1 promoter induced by IGF-1, and PI3K inhibitor and ERK inhibitor reduced the binding abilities of p65 to the FOXC1 promoter. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. The p values were obtained by one-way ANOVA (B, D, E, G, H, J, K,). Data are presented as mean (n = 3) ± S.D. All experiments were performed at least three times.