Figure 4. Misexpression of CITED1 both in human embryonic kidney cells (HEK293) that have been stably transfected (STF) with a TOPflash reporter and in WiT49 cells that had subsequent transient transfection of a TOPflash reporter.

(A) Empty vector control (pcDNA) shows baseline TOPflash activation in HEK293 cells bathed in L-cell conditioned medium (L CM). Wild-type CITED1 shows almost two-fold repression of TOPflash activation, whereas transfection of the CITED1ΔNES shows restored responsiveness. (B) HEK293 cells stably transfected with a TOPflash plasmid and grown in the presence of Wnt3a CM showed marked reporter assay activation, as estimated from the empty vector controls (pcDNA). Transfection of wild-type CITED1 in these HEK293 cells showed more than two-fold repression of Wnt3a activation, whereas transfection with the CITED1ΔNES overcame much of this repressive function. Experiments for A and B were completed in triplicate and repeated on consecutive weeks. (C) We observed an identical effect of these CITED1 constructs when stably transfected into malignant WiT49 cells and then subsequently transiently transfected with TOPflash. Four biological replicates were performed with different passages (4–7), and one passage was repeated (4a and 4b). Regardless of passage number, overexpression of wild-type CITED1 in WiT49 cells repressed Wnt3a activation, whereas the CITED1ΔNES again appeared to overcome this repressive effect. Notably, in our model, wild-type CITED1 in nearly exclusively cytosolic in both HEK293 and WiT49 cells, but the CITED1ΔNES shows nearly evenly distributed subcellular localization between the cytosol and nucleus, which may reflect a mixed response effect.