JCS007492 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 (Adobe PDF) -

  Fig. S1. (A) Relative expression levels of PML proteins. Western blot for PML expression in primary MEF Pml^−/− cells transduced with the different isoforms. (B) Typical localization patterns of PML isoforms in transduced MEF cells. Note that none of them colocalises with B23 in untreated conditions. (C) Primary Pml^−/− MEFs were treated with 5 μM MG132 for 15 hours. Note that all isoforms relocalise to de novo structures in close contact to B23-labelled structures after disruption of nucleolar organization by proteasome inhibition. (D) PML-I, -IV and -V form large, hollow nuclear bodies that contain B23 6 hours after γ-radiation (5 Gy). (E) PML-I and -IV are efficiently targeted to the nucleolar periphery 15 hours after doxorubicin treatment (2 μg/ml). (F) PML relocalises to the nucleoplasm following UVC treatment (35 J/m²) in an isoform-independent manner. (G) Deletion of the C-terminal domain of PML-I abolishes nucleolar targeting under stress. Confocal images of MEF Pml^−/− cells transduced with either PML-I or a C-terminus deletion construct lacking amino acids 553-882 (PML-I ΔC), treated or not with 0.1 μg/ml doxorubicin for 16 hours, as indicated.

• Supplemental Figure S2 (Adobe PDF) -

  Fig. S2. (A) Multiple alignment of PML-I C-termini from different species using ClustalW. (B) Computer prediction of an exonuclease-III-like motif in the C-terminus of PML-I. E=3.10-6 for human PML-I C-terminus with rpsblast, database Smart − 663PSSMs. The prediction was made using RPS-BLAST 2.2.11 and the database cdd.v2.05. Organism annotation: A (accession number 1J538A) chain A, structure of the N-terminal exonuclease of the epsilon subunit of E. coli DNA polymerase III at pH 8.5; B (acc. no. gi_2291256): hypothetical protein COSC8.5 (C. elegans); C (acc. no. gi_15240411): exonuclease (A. thaliana); D (acc. no. gi_1256855): Ylr107wp (S. cerevisiae); E (acc. no. gi_4929697): CGI_114 (H. sapiens); F (acc. no. gi_15643342): DNA polymerase III, α subunit (T. maritima); G (acc. no. gi_1706437): probable ATP-dependent helicase DinG homologue (B. subtilis); H (acc. no. gi_6014995): DNA polymerase III, ε subunit (A. aeolicus); I (acc. no. gi_21263566): DNA polymerase III, pol C-type (S. aureus).

• Supplemental Figure S3 (Adobe PDF) -

  Fig. S3. Comparison between human and chicken PML-I C-termini. Alignment was performed using Dotmatcher (matrix EPAM 50, window 9, threshold 99). The putative Exo-III domain, as well as amino acid residues are indicated.

• Supplemental Figure S4 (Adobe PDF) -

  Fig. S4. (A-C) Discrete (A) Sp100, (B) SUMO or (C) ubiquitin domains can aggregate onto SANB.

• Movie 1 -

  Movie 1. Video-microscopy of WI-38 cells transduced with GFP-PML-I.