Fig. 7: MAFF binds to BACH1 to activate IL11-STAT3 pathways.

a MAFF binding proteins were immunoprecipitated using MAFF antibody and analyzed by mass spectrometry. In MDA-MB-231 cells, BACH1 was a main binding partner of MAFF. The graph indicates the unique peptide number identifying MAFF binding compared to IgG control. n = 3 biological replicates. Unpaired t-test. b Co-immunoprecipitation confirmed the binding of MAFF and BACH1, which was further increased under hypoxia. c When BACH1 was knocked down, mRNA expression of IL11 was decreased. n = 3 biological replicates. Unpaired t-test. d The downstream effect of BACH1 on the STAT3 pathway was determined by western blotting after BACH1 was knocked down in MDA-MB-231 both under normoxia and hypoxia. e ChIP-qPCR confirmed that MAFF and BACH1 bindings on the MARE sequence of IL11 promoter, which was identified from ChIP-sequencing. Bindings were further increased under hypoxia. Unpaired t-test. f Luciferase assay was performed using pGL4-luciferease vector, which was subcloned with the identified MARE sequence on IL11 promoter. While hypoxia increased the luciferase activity, BACH1 or MAFF knockdown decreased the transcriptional activity of the identified MARE. n = 3 biological replicates. Unpaired t-test. Knocking down BACH1 or both MAFF and BACH1 decreased tumor cell invasion through Matrigel coated transwells (g) and tube formation of HUVEC representing angiogenic features (h) (CM: cell media). However, IL11 overexpression rescued the reduction in cell invasion and tube formation. n = 3 biological replicates. One-Way ANOVA with multiple comparisons. Graphs represent the mean per group and error bars represent the SEM.