A) PML C/C-is not modified by SUMO in (i) U2OS, (ii) JFCF-6/T.1R and (iii) MCF7 cells. U2OS and JFCF-6/T.1R are established ALT cell types while MCF7 is a telomerase-positive cell line. The cells were transfected with the indicated plasmids. Cell lysates were harvested in the presence and absence of N-ethylmaleimide (NEM) 48 h after transfection and protein expression was detected with western blot analysis. Wild-type PML is HA-tagged while PML C/C- is FLAG-tagged. Blots were probed with the indicated antibodies. B-actin was used as a loading control. B) APBs are ALT-specific. Endogenous APBs were detected in (i) U2OS and (ii) JFCF-6/T.1R cells but not in (iii) MCF7 cells. PML antibodies were used to detect endogenous PML. ( C) Transiently over-expressed HA-tagged wild-type PML co-localized with TRF2 in (i) U2OS and (ii) JFCF-6/T.1R but not in non-ALT (iii) MCF7 cells. ( D) Transiently over-expressed FLAG-tagged coiled-coil domain deficient PML mutant did not form distinct nuclear bodies in all three cell-lines, (i) U2OS, (ii) JFCF-6/T.1R, (iii) MCF7. E) Graph depicting the percentage of cells expressing APBs. At least 100 cells were scored and the results were summarized from at least three independent experiments. Values represent the mean ± SD. APBs are defined by the co-localisation of PML with TRF2 and are indicated by arrows. PCI Neo was used as an empty vector control. DAPI was used for visualisation of the nucleus and the indicated antibodies were used. Images were captured with confocal microscopy at 100 X magnification. * indicates p < 0.05.