JCS02965 Supplementary Material

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• Supplemental Figure 1 -

  Fig. S1. (A,B) The ALT pathway is characterised by the colocalisation of PML nuclear bodies, telomere-associated protein TRF1 and TRF2, and telomeric DNA repeats (AATCCC)n. Our results demonstrated that, in EBV-transformed ICF cells, the ALT pathway is not activated, in contrast to what observed in SV40-transformed WI-38 fibroblasts. (A) In ICF cells, TRF2 is not accumulated in the giant ICF body. (B) In SV40-transformed WI-38 fibroblasts, TRF2 accumulates within ALT-PML bodies.

• Supplemental Figure 2 -

  Fig. S2. In the giant ICF body, protein signals are round shaped. Either they have a pale central core (PML, BLM, TOPOIIIα, SUMO-1, CBP, SP100) or they are homogeneously stained (HP1s). These different patterns can be observed by decomposition into colours and enlargement (×1.6) of the protein signals colocalising in the giant body.

• Supplemental Figure 3 -

  Fig. S3. The non-transformed PHA-stimulated peripheral lymphocytes of ICF patients also show a giant PML body, at the G2 phase. Decomposition into colours and enlargement (×1.6) of protein signals colocalising within the giant body allowed us to confirm the presence of two types of protein patterns. These patterns, similar to those observed in EBV-transformed cell lines, support a similar protein organisation of the giant bodies.

• Supplemental Figure 4 -

  Fig. S4. At the G2 phase, EBV-transformed cell lines of control subjects show PML-NBs that are extremely variable in size. In the larger PML-NBs (arrow), decomposition into colours and enlargement (×1.6) allow us to confirm the presence of two types of round-shaped protein patterns. Such results suggest that the giant ICF body and the PML-NBs share a similar organisation with proteins arranged in spheric concentric layers.

• Supplemental Figure 5 -

  Fig. S5. The NB4 cell line is derived from an acute promyelocytic leukaemia patient carrying a t(15;17) translocation that generates a fusion protein between PML and RARα. The fusion protein exerts a dominant-negative effect on wild-type PML, resulting in the loss of PML function in NB4 cells (Zhu et al., 1997). Using NB4 cells synchronised at the G2 phase, we performed immuno-FISH experiments with several PML-NB-associated proteins and a 1qh satellite (1qh sat.) DNA probe. We demonstrated that none of the proteins PML, DAXX, ATRX, BLM, TOPOIIIα or HP1α was able to form a large round-shaped body colocalising with 1qh satellite. Each box shows the same NB4 nucleus with the red protein pattern (left) and the green 1qh satellite signals (right). In these nuclei, three 1qh satellite signals are detected, which is in agreement with the hypertriploid chromosome number of the NB4 cell line (Mozziconacci et al., 2002).