Cytoplasmic sequestration of HIPK2 in filamentous structures formed by interaction between RUNX1 and PEBP2-β-SMMHC. (A) PEBP2-β-SMMHC forms filament-like cytoplasmic structures in the presence of RUNX1. COS-7 cells were transiently transfected with RUNX1 along with PEBP2-β or PEBP2-β-SMMHC and analyzed for RUNX1 (green) and PEBP2-β or PEBP2-β-SMMHC (red) by indirect immunofluorescence with anti-RUNX1 or -PEBP2-β antibodies accordingly, followed by Alex 488- or 594-conjugated secondary antibodies, respectively. Merged images showing overlapping localization are shown in the third panel. The nuclei were counterstained with DAPI (blue). All images: original magnification ×1000. (B) HIPK2 is specifically sequestrated in pronounced cytoplasmic RUNX1/PEBP2-β-SMMHC complexes. FLAG-HIPK2 is coexpressed together with wild-type PEBP2-β and RUNX1 (first column), β-SMMHC alone (second column), β-SMMHC and RUNX1 (third column), in COS-7 cells. Negative control experiment was performed using FLAG-DYRK1A (fourth column). FLAG-HIPK2 (green), FLAG-DYRK1A (green), PEBP2-β (red), or β-SMMHC (red) proteins were visualized by indirect immunofluorescence as described for panel A using anti-FLAG or -PEBP2-β antibodies, respectively. Merged images showing nuclei counterstained with DAPI (blue) are shown in the lowest panels. (C) Sequestration of HIPK2 to RUNX1/β-SMMHC complex is dependent on RUNX1 dimerization and not DNA-binding activity. FLAG-HIPK2 is coexpressed together with β-SMMHC and wild-type RUNX1 (first column), dimerization-defective RUNX1^S67R (second column), or DNA binding-defective RUNX1^K83E (third column) in COS-7 cells. Ectopically expressed β-SMMHC (red) and FLAG-HIPK2 (green) were visualized by indirect immunofluorescence, and in merged images nuclei were counterstained with DAPI (blue). (D) U937 cells coexpressing ectopic FLAG-HIPK2 and β-SMMHC proteins were stained and visualized by indirect immunofluorescence microscopy to reveal the sequestration of HIPK2 to prominent cytoplasmic β-SMMHC filamentous structures.