Fig. 6. AFF4 ablation in MCF7 cells inhibits ERα expression and cell growth.

a AFF4 was knocked out in MCF7 cells using CRISPR/Cas9 technology. Cell lysate from a control and three different AFF4-knockout clones was extracted and subjected to western blot analysis to determine the expression levels of AFF4 and ERα. b Knockout of AFF4 reduced the growth of MCF7 cells in culture medium containing regular FBS. c Knockout of AFF4 reduced the growth of MCF7 cells in culture medium with charcoal-stripped FBS. d AFF4 knockout reduced the recruitment of Cyclin T1 and RNA polymerase II to ESR1 gene. Chromatin prepared from the control or AFF4-knockout MCF7 cells was used for ChIP-qPCR. Primers for TSS region of ESR1 gene were used for qPCR. Cells were cultured in medium containing charcoal-stripped FBS. e Amino acid F1103 of AFF4 is critical for AFF4 to interact with H3K27ac. 293 T cells were transfected with empty vector or different AFF4 mutants, and the cell lysate was collected for H3K27ac peptide pull-down assay. f Amino acid F1103 of AFF4 is essential for ERα expression. AFF4-knockout MCF7 cells were transfected with empty vector or different AFF4 mutants, and western blot assay was performed to determine the protein levels of AFF4 and ERα. Cells were cultured in medium containing charcoal-stripped FBS. The error bars were shown as SD; n = 5 (b, c) or 3 (d) biological replicates; P-value was determined by two-tailed Student’s t-test.