Fig 1: IGF2 facilitates BACH1 upregulation via the ERK1/2/ETS1 pathway. (A-B) The mRNA and protein level of BACH1 when PLC/PRF/5 (A-left, B-upper) and HepG2 cells (A-right, B-lower) were treated with IGF2 at the 0 ng/ml, 25 ng/ml, 50 ng/ml, and 100 ng/ml for 24 h. (C) The relative luciferase activities of BACH1 promoter reporter vectors in PLC/PRF/5 cells (left) and HepG2 cells (right) treated with IGF2 (100 ng/ml) for 24 h were analyzed by luciferase reporter assays. (D) The protein levels of BACH1, p-AKT, AKT, p-ERK1/2, and ERK1/2 in PLC/PRF/5 cells treated with PI3K inhibitor (LY294002, 20 μM) or ERK inhibitor (SCH772984, 100 nM) in the presence of IGF2 (100 ng/ml, 24 h). (E) The serially truncated/mutated BACH1 luciferase reporter activities in the PLC/PRF/5 cells treated with or without IGF2 (100 ng/ml, 24 h). (F-H) The PLC/PRF/5 cells were transfected with shETS1 or control shRNA in the presence of IGF2 (100 ng/ml, 24 h). The mRNA expression of BACH1 was detected by RT-qPCR (F). The protein levels of BACH1, ETS1 and p-ETS1 were detected by western blot (G). The relative luciferase activities of BACH1 promoter reporter vectors were analyzed by luciferase reporter assays (H). (I) The effects of ERK inhibitor on BACH1, p-ERK1/2, ERK1/2, p-ETS1, and ETS1 levels in PLC/PRF/5 cells treated with IGF2 (100 ng/ml, 24 h). (J) ChIP analysis of ETS1 binding to the BACH1 promoter region in PLC/PRF/5 cells treated with PI3K inhibitor or ERK inhibitor in the presence of IGF2 (100 ng/ml, 24 h). *p < 0.05, **p < 0.01, ***p < 0.001, NS: no statistical difference. Data were shown as Mean ± SD.
Fig 2: IGF1R blockade combined with PTK2 inhibition suppresses BACH1-mediated HCC growth and metastasis. (A) The protein levels of BACH1, p-IGF1R, IGF1R, p-PTK2, PTK2 in PLC/PRF/5-LV-BACH1 cells treated with linsitinib alone or defactinib alone or a combination of both. (B-C) Transwell assay was performed to analyze the migratory and invasive capacity of PLC/PRF/5-LV-BACH1 cells treated with linsitinib alone or defactinib alone or a combination of both. Scale bar, 100 µm. (D) Schematic diagram of drug treatment for nude mice. (E-J) HCC orthotopic xenograft models indicated that the combination of linsitinib and defactinib suppressed BACH1-mediated HCC growth and metastasis. The representative bioluminescent imaging in the liver (E), the bioluminescent signals of liver tumors (F), the occurrence of pulmonary metastasis (G), the representative H&E staining images of pulmonary tissues (H), the number of pulmonary metastasis lesions (I) from nude mice after orthotopic transplantation with PLC/PRF/5-LV-BACH1 cells treated with linsitinib alone or defactinib alone or a combination of both were shown. The OS of mice was analyzed in (J). Scale bars, 2 mm (upper), 200 µm (lower). *p < 0.05, **p < 0.01, ***p < 0.001. Data were shown as Mean ± SD. (K) A schematic diagram showing how BACH1 facilitates HCC growth and metastasis and the combination strategy for HCC. BACH1 upregulates IGF1R and PTK2 to promote HCC growth and metastasis. IGF2, the ligand of IGF1R, in turn upregulates BACH1 expression through the IGF1R-ERK1/2-ETS1 cascades, thus forming a positive feedback loop to stimulate HCC cells continuously. IGF1R blockade combined with PTK2 inhibition significantly suppresses BACH1-mediated HCC malignant progression.
Fig 3: IGF2 promotes HCC growth and metastasis depending on BACH1. (A) BACH1 was silenced in PLC/PRF/5 cells. IGF1R and PTK2 were overexpressed in BACH1-silencing PLC/PRF/5 cells. These cells were treated with IGF2 at 100 ng/ml for 24 h. The protein levels of BACH1, p-IGF1R, IGF1R, p-PTK2 and PTK2 in the indicated PLC/PRF/5 cells were detected by western blot. (B-C) The migratory and invasive capacity of the indicated PLC/PRF/5 cells were analyzed by transwell assay. Scale bar, 100 µm. (D) IGF2 was overexpressed in PLC/PRF/5 cells and then BACH1 was silenced in IGF2-overexpressing PLC/PRF/5 cells. IGF1R and PTK2 were overexpressed in BACH1-silencing PLC/PRF/5-LV-IGF2 cells. The protein levels of IGF2, BACH1, p-IGF1R, IGF1R, p-PTK2 and PTK2 in the indicated PLC/PRF/5 cells were detected by western blot. (E-F) The migratory and invasive capacity of the indicated PLC/PRF/5 cells were detected by transwell assay. Scale bar, 100 µm. (G-L) The indicated HCC cells were used to construct HCC orthotopic xenograft models. The representative bioluminescent imaging in the liver (G), the bioluminescent signals of liver tumors (H), the occurrence of pulmonary metastasis (I), the representative H&E staining images of pulmonary tissues (J), the number of pulmonary metastasis lesions (K) from nude mice after orthotopic transplantation with indicated PLC/PRF/5 cells were shown. The OS of mice was analyzed in (L). Scale bars, 2 mm (upper), 200 µm (lower). *p < 0.05, **p < 0.01, ***p < 0.001. Data were shown as Mean ± SD.
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