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Fig. 1. ANKRD11 associates with p14^ARF, p53 and PML in extranucleolar inclusions. (A) GFP-ANKRD11 localizes to subnuclear domains that are visible using phase-contrast microscopy (upper panels). MCF-10A cells were transduced with a recombinant retrovirus expressing GFP-ANKRD11, selected in geneticin and imaged using confocal microscopy. MCF-10A cells stably expressing GFP-ANKRD11 were immunostained with anti-nucleophosmin antibody (nucleolar marker; red) and DAPI (4',6-diamidino-2-phenylindole; DNA; blue) (lower panels). (B) Endogenous ANKRD11 colocalizes with p53. MCF-10A cells were either untreated or treated with mitomycin C (20 µg/ml) for 6 hours and immunostained with anti-ANKRD11 and anti-p53 (Ab-5) antibodies. The relative p53 protein levels in these cells with up to 6 hours of mitomycin C treatment was determined by western blot analysis using an anti-p53 antibody. β-actin was used as a loading control. (C) ANKRD11-myc localized exclusively to p14^ARF-positive extranucleolar inclusions. HEK293T cells were transiently transfected with constructs expressing GFP-p14^ARF and ANKRD11-myc. GFP-p14^ARF localized to both nucleoli (hollow arrowheads) and extranucleolar inclusions (filled arrowheads). Localization of ANKRD11-myc was determined through immunostaining with anti-myc antibody. (D) Endogenous PML colocalizes with p14^ARF and ANKRD11-myc in extranucleolar inclusions. HEK293T cells from C, ectopically expressing GFP-p14^ARF and ANKRD11-myc, were immunostained with anti-myc and anti-PML antibodies.

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