Activation of the WNT pathway after BORIS over-expression. (A) Western blot analysis of WNT5A/B and TCF3 in untransfected control, mock transfected (C3-empty) or BORIS transfected (C3-BORIS) HEK293T cells. The upper band in the WNT5A/B blot is non-specific (*) as shown by provider (Cell Signalling). The membranes were probed with the antibodies as indicated and GAPDH was used as a loading control. Lysates from three independent experiments were loaded on the same gel. (B) mRNA levels of BORIS bound transcripts in HEK293T cells over-expressing BORIS (C3-BORIS) as compared to empty vector (C3-empty). Data in (B) represent 2 technical replicates ± SD. (C) Luciferase activity of a TCF/LEF-responsive element reporter after BORIS (C3-BORIS) or mock transfection of HEK293T cells. Over-expression of BORIS (C3-BORIS) led to a more than four-fold increase in luciferase activity compared to cells transfected with empty vector alone (C3-empty). Cignal negative is a non-inducible reporter, lacking the TCF/LEF response element. LiCl was used as a general activator of the TCF/LEF reporter. Data are expressed as a mean ratio of the Firefly/Renilla luciferase activity (see Materials and Methods) and represents the average ± SD of 4 biological replicates. (D) Western blot analysis of HEK293T cells after over-expression of BORIS together with siRNA knock down of β-catenin. LiCl treated cells (C3-empty + LiCl) were included in the analysis to exclude a direct effect of LiCl on the expression of β-catenin. GAPDH was used as a loading control. (E) Densitometry quantification of β-catenin expression in (D). Data represents the mean ± SD of 3 biological replicates. (F) Normalised TCF/LEF luciferase reporter activity after β-catenin knock-down and BORIS over-expression in HEK293T cells. β-catenin knock-down efficiently prevented BORIS-mediated activation of the TCF/LEF luciferase reporter, compared to Scrambled + Reporter with β-catenin siRNA + Reporter. Data shown is from one representative experiment.