The formation of APBs is independent of the SUMOylation of the PML protein. All PML KR mutants can form APBs in ALT cells. A) U2OS and B) JFCF-6/T.1R cells were transfected with (i) empty vector PCI Neo, (ii) wild-type PML, (iii) PML K65R, (iv) PML K160R, (v) PML K490R, (vi) PML K65/160R, (vii) PML K65/490R, (viii) PML K160/490R and (ix) PML triple K65/160/490R plasmids. (x) Graphs depicting percentage of cells expressing APBs. At least 50 cells were scored and the results were summarised from at least three independent experiments. Values represent the mean ± SD. All PML plasmids are HA-tagged. In PCI Neo transfected cells (i), detection of APBs was based on co-localisation of endogenous PML and TRF2. Arrows indicate detection of APBs, defined as the co-localisation of PML with TRF2. Immunofluorescence was performed 48 h after transfection and the indicated antibodies were used. DAPI was used for nuclear staining. Images were captured with confocal microscopy at 100 X magnification. * indicates p < 0.05.