Figure 5.

TAK1 enhances TGF-β1–induced phosphorylation of p38 and apoptosis in PC-3U cells. PC-3U cells were transfected with either wild-type HA-TAK1 or kinase inactive HA-TAK1-K63W and treated with TGF-β1 for 12 or 24 h. (A) Coimmunofluorescence stainings for HA, detected by fluorescein isothiocyanate and phosphorylated p38 (phospho-p38) by tetramethylrhodamine B isothiocyanate. An overlay of both pictures with additional stainings of nuclei with DAPI (merge + DAPI) reveals a high degree of colocalization of HA-TAK1 and phospho-p38 in PC-3U cells expressing wild-type HA-TAK1, both before and after TGF-β treatment for 12 h. In contrast, PC-3U cells expressing HA-TAK1-K63W, do not express phospho-p38 after TGF-β1 treatment, as shown in the overlay of both pictures with additional stainings of nuclei with DAPI (merge + DAPI). (B) Coimmunofluorescence stainings for endogenous TAK1 (by using polyclonal antibodies against TAK1) as shown by tetramethylrhodamine B isothiocyanate and the apoptotic marker M30, as shown by fluorescein isothiocyanate, was used to identify apoptotic cells after TGF-β treatment for 24 h. An overlay of both pictures with additional stainings of nuclei with DAPI (merge + DAPI) reveals a high degree of colocalization of TAK1 and M30, in PC-3U cells expressing wild-type HA-TAK1. In contrast, in PC-3U cells expressing HA-TAK1-K63W, no colocalization of mutant TAK1 and M30 was observed, as shown in the overlay of both pictures with additional stainings of nuclei with DAPI (merge + DAPI). The number of transfected cells, expressing high levels of TAK1, and the number of M30-positive cells, were counted in several, randomly chosen fields. At total of at least 1000 cells was counted. The ratio between M30-positive cells and transfected cells is shown below the picture.