Fig 2: SUMO5 conjugates PML as polymeric chains.(A) SUMO5 can conjugate PML. GFP-tagged SUMO5 was co-expressed with Flag-tagged PML-I, PML-IV, or PML-VI in 293 cells. Anti-Flag immunoprecipitates were immunoblotted with anti-Flag or anti-GFP antibodies as indicated. (B) SUMO5(I95R), which retains the di-glycine motif (G96G97) after tryptic digest, is used to identify peptide fragments conjugated by SUMO5 in LC-MS/MS analyses. (C) MS/MS confirms that SUMO5 conjugates PML at K160, K380, K400, K490, K497, and K490/K497. Anti-Flag immunoprecipitates from 293 cell lysates expressing Flag-PML-IV and GFP-SUMO5(I95R) were separated by SDS-PAGE and analyzed by LC-MS/MS. (D) SUMO5 conjugates PML at K65, K160, and K490. Flag-tagged SUMO5 was co-expressed with GFP-tagged wild-type or mutant PML. Anti-Flag immunoprecipitates were immunoblotted with anti-GFP or anti-Flag antibodies. PML(3K), mutant PML with lysines 65, 160, and 490 mutated to arginines. Bracket, SUMO-modified PML. Arrowhead, unmodified PML. +1 next to an arrowhead indicates the position of PML conjugated by one SUMO5 molecule. (E) SUMO5 poly-SUMOylates PML. GFP-tagged SUMO5 or mutants were co-expressed with HA-tagged PML in 293 cells. Anti-HA immunoprecipitates were immunoblotted with anti-HA or anti-GFP antibodies as indicated. Bracket, SUMO-modified PML. Arrowhead, unmodified PML.

Fig 3: ZBTB48 binds telomeres via its ZnF11 domain in vitro and in vivo Domain structure of wild-type (WT) ZBTB48 containing a N-terminal BTB domain and 11 zinc fingers (ZnF) at the C-terminus of the 688-amino-acid protein. Note that ZnF2 is degenerate. Below is a schematic of the deletion construct lacking ZnF1-10 used in (B).Sequence-specific DNA pull-downs with either telomeric (TTAGGG) or a control sequence (GTGAGT) for FLAG-ZBTB48 WT, point mutants for the ten functional zinc fingers and a domain deletion construct for ZnF1-10.Sequence-specific DNA pull-downs for FLAG-ZBTB48 WT and ZnF11 point mutant for telomeric and subtelomeric variant repeat sequences (green) and their respective scrambled controls (blue).Co-localization analysis of endogenous ZBTB48 and TRF2 in U2OS cells by immunofluorescence (IF) staining. A representative image illustrating the co-localization between ZBTB48 (green) and TRF2 (red) as a marker for telomeres is shown with DAPI (blue) used as a nuclear counterstain. The quantification of frequency of co-localization events (right) was done after 3D reconstruction of the acquired z-stacks (n = 104 cells). The average value is indicated by a red bar.IF stainings for exogenous FLAG-ZBTB48 WT and point mutants for ZnF10 and ZnF11 in U2OS cells. The same analysis as in (D) was performed and average co-localization frequencies are shown (n = 24–33 cells). Error bars indicate standard deviations, and P-values are based on Student's t-test.Co-localization analysis between ZBTB48 (green) and PML (red) as a marker for PML bodies analogous to (D) in U2OS cells (n = 102 cells).Data information: (D–F) Co-localization events are indicated by white arrows. Scale bars represent 5 µm.Source data are available online for this figure.

Fig 4: SOX9 is critical for the regulation of breast cancer-initiating capacity downstream PML.(a,b) Effect of SOX9 silencing with two shRNA (sh9.1 and sh9.2) on SOX9 protein expression (representative western blot out of three independent experiments) (a) and on OSI formation (n=3) (b) in MDA-MB-231 cells. (c) Limiting dilution experiment after xenotransplantation. Nude mice were inoculated with 500,000 or 50,000 MDA-MB-231 cells (n=8 injections per experimental condition). Tumour-initiating cell number was calculated using the ELDA platform. A log-fraction plot of the limiting dilution model fitted to the data is presented. The slope of the line is the log-active cell fraction (solid lines: mean; dotted lines: 95% confidence interval; circles: values obtained in each cell dilution). (d,e) Effect of ectopic SOX9 expression on the consequences of PML silencing. Representative western blot (out of three independent experiments) depicting expression of PML and SOX9 (endogenous and ectopic protein are detected) (d) and OSI formation (n=3) (e) in the different experimental conditions in MDA-MB-231 cells. (f) Limiting dilution experiment to assess frequency of tumour-initiating cells after xenotransplantation. Nude mice were inoculated either with 500,000 or 50,000 MDA-MB-231 cells (n=12 per experimental condition, except in shC/Mock and sh4/SOX9, n=16). Tumour-initiating cell number was calculated using the ELDA platform as in c. (g) OSI formation in MDA-MB-231 cells transduced with the indicated constructs (mock, SOX9) and treated with vehicle or 150 nM ATO (n=6). (h) Diagram of the molecular mechanism by which PML controls the expression of the stem cell factor SOX9 to regulate BCa-initiating cell function. Error bars represent s.e.m., P value (*P<0.05; **P<0.01; ***P<0.001 compared with its control or as indicated). Statistic test: one-tail unpaired t-test (b,e,g) and ?2-test (c,f). ATO, arsenic trioxide; OSI, primary oncospheres; shC, Scramble shRNA; sh9.1 and sh9.2, shRNA against SOX9; sh4, shRNA against PML; VC, vehicle control.

Fig 5: PML-NBs potentially affect the efficiency of the response of SUMO upon exposure to proteotoxic stress. (A–C) Prolonged exposure to proteasome inhibitors. Representative images of the nuclei of maturing oocytes subjected to treatment with 10 µM MG132 for 39 h (A,B), or with 1 µM epoxomicin for 45 h (C, Epox.). Oocytes were stained with anti-splicing component, 35 kDa (SC35) (red) and anti-SUMO1 (green) (A) or Alexa 488-conjugated anti-SUMO2/3 (green) (B), or with anti-SUMO1 (green) only (C, z-stack). BF, bright-field image of the oocyte. (D) The response of representative PML clients to proteasome inhibition. Representative images of the nuclei of maturing oocytes left untreated (cont) or treated with 10 µM MG132 for 45 h. Oocytes were stained with anti-SC35 (green) and anti-ATRX or DAXX (red) antibodies. Scale bars: 4.98 µm. (E) The response of SC35-positive compartments to transcriptional inhibition. Representative image of the nucleus of maturing oocytes treated with dimethyl sulfoxide (DMSO, vehicle control) or 1 µM actinomycin D (AcD) for 16 h. Oocytes were stained with anti-SC35 (red) and anti-Ubc9 (green) antibodies. Scale bars: 2.00 µm. (F) Representative images of the nuclei of maturing oocytes treated with DMSO or 10 µM MG132 for the indicated time. Oocytes were stained with anti-Ubc9 antibody. (G) An examination of whether exogenously assembled PML-NBs alter the response of SUMO to proteotoxic stress. Representative image of the nucleus of maturing oocytes injected with a plasmid encoding GFP-hPMLVI and cultured for 25 h followed by treatment with 10 µM MG132 for 37 h. Oocytes were stained with anti-SUMO1 (magenta) and anti-SC35 (red) antibodies. Scale bars: 2.00 µm.