Human C28/I2 chondrocyte cells were cotransfected with either the empty pGL4 plasmid (luciferase reporter plasmid) or the pGL4 plasmids containing different regions of the PITX1 promoter combined with either the empty pBabe plasmid or pBabe plasmids expressing ER (estrogen receptor) fused to E2F1, E2F2, or E2F3 and induced with 4OH-tamoxifen (OHT) for 24 h. (A) The -3895/+61 PITX1 gene region contains regulatory elements capable of producing a luciferase signal that is not affected by OHT treatment. (B) Overexpression of E2F1, E2F2, and E2F3 produces a significant increase in the luciferase activity under the control of the -3895/+61 PITX1 gene region. (C) The proximal sequence of the PITX1 promoter contains several E2F1 binding sites, as predicted by MatInspector 8.0 software (Genomatix Software Suite). (D) Overexpression of E2F1 has variable effects on luciferase activity depending on the length of the transfected promoter region (-3895/+61; -374/+61; -279/+61; -214/+61; -169/+61; -84/+61). Except for the -84/+61 PITX1 gene region, all the other constructs are significantly activated by E2F1. (Fig 1A, 1B and 1D) Data represents mean and standard deviation of 3 independent experiments. Asterisks represent a significant increase in luciferase activity (Two-way ANOVA; Bonferroni post hoc: *p < 0.0001) compared with control cells.

https://doi.org/10.1371/journal.pone.0167530.g001