Fig 3. MEF2C and EBF1 can functionally co-regulate their common targets.

(A) Relative luciferase activities of either WT pGL4.23-Il7ra (black bars) or pGL4.23-Il7ra containing mutated MEF2C binding site (white bars) in 293T cell lysates expressing FLAG-tagged WT, EED, or AAA MEF2C, and/or Myc-tagged EBF1, as indicated on the x-axis. Renilla luciferase was used as internal control, and the experiments were performed in biological triplicates, each with technical triplicates; asterisk denotes p-value<0.5 and double-asterisk denotes p-value<0.01 (student t-test, one-tail, paired). (B) Relative luciferase activities of WT pGL4.23-Il7ra in 293T cell lysates expressing the same MEF2C constructs as (A), along with Myc-tagged E12 or E47; experiments were performed in technical triplicates. (C) Luciferase reporter activities of 293T cell lysates transfected with pGL4.23-trimerized EBF1 and MEF2C binding sites with either MEF2C or EBF1 alone or in combination. Cells were transfected with either WT (WT_3xE/M, black bars) construct or a construct with mutated EBF1 and MEF2C binding sites (Mutant_3xE/M, white bars); the mutations are indicated in black in the scheme; experiment was performed in technical triplicates. (D) Relative expression levels of Il7ra and Ebf1 in mouse lineage-depleted progenitor (lin-) cells that over-express either empty vector (EV), WT, or EED MEF2C, two days after transduction; summary of two biological duplicates is shown. (E) Relative expression levels of Il7ra and Pou2af1 in lin- cells that over-express either EV, WT MEF2C, EBF1 individually, or MEF2C and EBF1 incombination. Summary of two biological duplicates is shown.