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1 DOI: 1.138/ncb37 a Supplementary Figure 1 Dapi EU γh2ax Overlay b Dapi NBS1-GFP Firbillarin Overlay c Pre-laser 2 min 5 min 1 min 2 min 3 min 4 min 5 min 6 min 7 min d Dapi GFP-MRE11 γh2ax Overlay Supplementary Figure 1 Additional data on rrna silencing after DNA damage and NBS1 and MRE11 nucleolar localization. (a) Measurement of rrna synthesis after laser-irradiation by 5-EU incorporation. A larger field of cells is shown here as compared to Figure 1A (2 independent experiments; one representative image shown). (b) U2OS cells stably transfected with NBS1-GFP were presensitized with 1uM BrdU for 24 h and exposed to laser micro-irradiation. To mark nucleoli, cells were immunostained with antibodies against Fibrillarin (2 independent experiments; 1 representative experiment shown). (c) Translocation of NBS1-GFP was followed by timelapse microscopy (5 independent experiments; 1 representative experiment shown). (d) U2OS cells stably transfected with GFP-MRE11 were presensitized with 1 µm BrdU for 24 h and exposed to laser micro-irradiation. Cells were immunostained with antibodies against gh2ax (2 independent experiments; 1 representative experiment shown). Scale bars: 1 µm. 1

2 a 1.2 EU Incorporation (Rel. values) b IR (Gy) 12 Intensity (EU594) simre11-59 simre11-6 NBS1 2 MRE11 simre11-59 simre Gy simre Gy simre Gy Supplementary Figure 2 Measurement of rrna synthesis after IR by EU labeling. (a) Dose titration of rrna synthesis after IR. Error bars represent S.E.M. (n = 3 independent experiments). (b) rrna synthesis after IR upon depletion of MRE11 by two different sirnas (measured by EU labeling 2 min after 5 Gy). The bars in the graph represent the average of 2 independent experiments. 2

3 a 1.2 DMSO Pre-rRNA (Rel. units) ATMi.2 Mock 1 Gy CPT b sinbs1-1 Pre-rRNA (Rel. units) sinbs1-2 sinbs1-1 sinbs1-2 NBS1 Tubulin c Mock 1 Gy CPT Pre-rRNA (Rel. units) simdc1 simdc1 MDC1 Tubulin.2 Mock 1 Gy CPT Supplementary Figure 3 Measurement of rrna synthesis after IR and CPT treatment by qrt-pcr. (a) rrna synthesis after IR and CPT treatment in the presence of ATM inhibitor (KU55933). Error bars represent S.E.M. (n = 4 independent experiments; samples were run in duplicates). (b) rrna synthesis after IR and CPT treatment upon depletion of NBS1 by two different sirnas. Error bars represent S.E.M. (n = 4 independent experiments for sinbs1-1; n = 3 independent experiments for sinbs1-2; all samples were run in duplicates). (c) rrna synthesis after IR and CPT treatment upon depletion of MDC1. Error bars represent S.E.M. (n = 4 independent experiments; samples were run in duplicates). 3

4 N-term Repeat region C-term NH2 COOH T-1: T-2: T-3: 3 56 T-4: T-5: T-6: T-7: T-8: Mw/kD GST T-1 MDC1 M-4 MDC1 M-3 T-3 T-2 GST-Treacle T-4 T-5 T-6 T-7 T-8 Autoradiograph Coomassie blue Supplementary Figure 4 Treacle is heavily phosphorylated by CK2 in vitro. Bacterially purified GST-Treacle fragments were incubated with recombinant CK2 and γ[ 32 P]-ATP, followed by SDS-PAGE and autoradiography. MDC1 fragments were used as positive and negative control, respectively 6. Note that fragment T-2 (aa 19-33) that contains the NBS1-interacting region is efficiently phosphorylated by CK2 in vitro (2 independent experiments; 1 representative experiment shown). 4

5 a Dapi Treacle UBF b Arb. units H H1 H18 GAPDH IgG mock IgG IR GFP mock GFP IR c Relative Intensity (arb. units) Time (s) Supplementary Figure 5 Treacle localization and mobility in the nucleoli. (a) Hela cells were immunostained with antibodies against Treacle and UBF (2 independent experiments; 1 representative experiment shown). Scale bar: 1 µm. (b) ChIP experiment with antibodies against mouse IgG or GFP in stably transfected U2OS GFP-Treacle cells. GAPDH was used as an unspecific target. Error bars represent S.E.M. (n = 4 independent experiments). (c) FRAP experiment in GFP-Treacle-expressing Hela cells. Bleach pulse was directed against one single nucleolus and fluorescence recovery was measured in the same nucleolus. T 1/2 = 21 ± 4 sec. The black line represents the median of 17 FRAP experiments included in the graph. 5

6 a N-term Repeat region NH2 COOH b Dapi GFP-Treacle-c γh2ax Overlay sinbs1 trol trol sinbs1 NBS1 Ponceau c NBS1-GFP-positive nucleoli (fold increase / background Mock PARPi Supplementary Figure 6 Additional data about Treacle localization to sites of DNA damage. (a) Schematic representation of Treacle-c. Note that the C-terminal region comprising the nucleolar localization signal is missing from this variant. (b) U2OS cells stably expressing GFP-Treacle-c were pre-sensitized with 1 µm BrdU for 24 h and exposed to laser microirradiation. Cells were treated with control sirna and sirna against NBS1 and immunostained with antibodies against gh2ax (2 independent experiments; 1 representative experiment shown). Scale bar: 1 µm (c) U2OS cells stably transfected with NBS1-GFP were pre-treated with PARP inhibitors and exposed to laser micro-irradiation. NBS1-GFP positive nucleoli were quantified. Error bars represent S.E.M. (n = 3 independent experiments; at least 45 cells with laser stripes probed for nucleolar NBS1 foci in each experiment, for statistical source data see Supplementary Table 2). 6

7 ATMi ++ IR siluc + + sinbs1 + + HA-Treacle HA-Treacle INPUT NBS1 SMC1 psq (ps61 Bid) HA-IP HA-Treacle NBS1 Supplementary Figure 7 Treacle phosphorylation in response to DNA damage. 293T cells were treated with the indicated sirnas for 72h and subsequently transiently transfected with HA-Treacle as indicated. 1h prior to irradiation indicated samples were treated with 1 µm ATMi (KU55933). Cells were exposed to 1 Gy of IR and immunoprecipitation was performed against HA (Treacle). Blots were probed with the indicated antibodies, including an antibody against psq (ps61 Bid; 2 independent experiments; 1 representative experiment shown). 7

8 Supplementary Figure 8 Figure 4 4c 4d (left) 4d (right) 5 4e 4f Figure c 5b (upper left) 5b (upper right) b (lower right) 5b (lower left) Figure 6 6a (left) 6a (right) Supplementary Figure 8 Full scans 8

9 Supplementary Table 1 Primer and sirna sequences sirna sequences (5 à 3 ) Treacle Treacle shrna ATM (used in combination) ATM (used in combination) NBS1 #1 NBS1 #2 MDC1 Control (Luciferase) CCACCAUGGGUUGGAACUAAAUU-dTdT CCACCATGGGTTGGAACTA TATATCACCTGTTTGTTAG- dtdt AGGAGGAGCTTGGGCCTTT- dtdt GGAGGAAGAUGUCAAUGU UTT- dtdt CCAACUAAAUUGCCA AGUATT- dtdt GUCUCCCAGAAGACAGUGA- dtdt CGUACGCGGAAUACUUCGAT-dTdT Treacle site-directed mutagenesis primer (5 à 3 ) S171A CAAATACT CGTTGGTCGCAGAAACTGAGGAGGAG T173A CGTTGGTCTCAGAAGCTGAGGAGGAGGGC T23A CAGCTCCAGCGAGGACGCCTCCAGCTCCAGTGATG T21A CTCCAGCTCCAGTGATGAGGCAGACGTGGAGGGGAAACC Primers for pre-rrna qrt-pcr (5 à 3 ) pre- rrna forward CCGCGCTCTACCTTACCTAC pre- rrna reverse GAGCGACCAAAGGAACCATA GADDH forward CCCATGTTCGTCATGGGTGT GADDH reverse TGGTCATGAGTCCTTCCACGATA Primer sequences for GST-Treacle fragments (5 à 3 ) GST- frag1- forward CGGGGATCCATGGCCGAGGCCAGGAAGCGGCGGGAG GST- frag1- reverse GCCGAATTCTTTTTCTTTCATGCTTGATGGCAAGTC GST- frag2- forward CGGGGATCCAACTCCTCAGTCCTGGGGGCGGACTTG GST- frag2- reverse GCGCCCTCGAGGGTCTGGGAGGCTACAGCCCCTGCCTT GST- frag3- forward CGGGGATCCAGAGCTGCCTCAGCCCCTGCCAAGGGG GST- frag3- reverse GCCGAATTCCACCTCTCCATCACTGCTGTCTGATGA GST- frag4- forward CGGGGATCCCCTGCAACCCCCTCAGCCCAGGTGGGG GST- frag4- reverse GCCGAATTCCTGTGCTGGAGATGCAGCTGCTTCCTC GST- frag5- forward CGGGGATCCGTCAAGGGGTCCTTGGGGCAAGGGATC GST- frag5- reverse GCCGAATTCTCAAGAAGACTCCTGGTCCTGGGCAGA GST- frag6- forward CGGGGATCCGGGCCTTCGGCTGCCCAGGCAGGGAAG GST- frag6- reverse GCCGAATTCCTCACTGTCATCTGAGCTCTCAGGGGC GST- frag7- forward CGGGGATCCCCCCAGAGCACCTCCGTCCAGGCCAAAGG GST- frag7- reverse GCCGAATTCGGCCTCCCCACCTTCCCCGGCCCCCAG GST- frag8- forward GCCGAATTCTACAGTCTGCTCTGCTGTCTTCTTCTTTTTC GST- frag8- reverse CGGGGATCCGTGGACACCACCAAGGAGAGCAGCAGG Primer sequences for HA-Treacle fragments (5 à 3 ) HA- Treacle- frag1 forward CATGGTACCCTTGTACAGCTC HA- Treacle- frag1 reverse GTGGAGGGGAAACCCTCAG HA- Treacle- frag2 forward CATGGTACCCTTGTACAGCTC HA- Treacle- frag2 reverse GTGGTGACCATGCCCACTGCC HA- Treacle- frag3 forward GGGTGTCACATCCCCCAC HA- Treacle- frag3 reverse GTGGTGACCATGCCCACTGCC HA- Treacle- frag4 forward AGCCTGGGTCAGTGGGAG HA- Treacle- frag4 reverse TGATCTAGAGGGCCCGTTTAAAC HA- Treacle- frag5 forward GGGTGTCACATCCCCCAC HA- Treacle- frag5 reverse TGATCTAGAGGGCCCGTTTAAAC Primer sequences for ChIP analysis (5 à 3 ) H- reverse GACGACAGGTCGCCAGAGGA H- forward GGTATATCTTTCGCTCCGAG H1- reverse ACGTGCGCTCACCGAGAGCAG H1- forward GGCGGTTTGAGTGAGACGAGA H18- reverse GGAAGTTGTCTTCACGCCTGA H18- forward GTTGACGTACAGGGTGGACTG GAPDH- forward TACTAGCGGTTTTACGGGCG GAPDH- reverse TCGAACAGGAGGAGCAGAGAGCG Supplementary Table 1 Primer and sirna sequences Table containing primer sequences for site-directed mutagenesis, PCR, qrt-pcr and ChIP analysis; and sirna sequences used to downregulate Treacle, ATM, NBS1 and MDC1 expression. 9

10 Statistical source data Fig. 2c Exp 1 H H1 H18 GAPDH IgG Mock IgG 2 Gy Pol1 Mock Pol 1 2 Gy E- 5 Exp 2 H H1 H18 GAPDH IgG Mock IgG 2 Gy Pol1 Mock Pol 1 2 Gy Exp 3 H H1 H18 GAPDH IgG Mock IgG 2 Gy Pol1 Mock Pol 1 2 Gy Average H H1 H18 GAPDH IgG Mock IgG 2 Gy Pol1 Mock Pol 1 2 Gy St Error H H1 H18 GAPDH IgG Mock IgG 2 Gy Pol1 Mock Pol 1 2 Gy Fig. 2d Exp 1 Exp 2 Exp 3 Exp 4 Average st Dev St. Error Primers for H IgG mock E E- 5 IgG 2 Gy E E- 5 Nbs1 mock Nbs1 2 Gy Primers for H1 IgG mock IgG 2 Gy 2 Nbs1 mock E E- 5 Nbs1 2 Gy Primers for H18 IgG mock E IgG 2 Gy E- 5 Nbs1 mock Nbs1 2 Gy Primers for GAPDH promoter IgG mock E- 5 IgG 2 Gy E E- 5 Nbs1 mock Nbs1 2 Gy Fig. 8a Exp 1 Exp 2 Exp 3 Average SEM ShVect + AcG ShTreacle# ShVect + AcG ShTreacle# Fig. 8b Exp 1 Exp 2 Exp 3 Exp 4 Average SEM Mock ActD Suppl. Fig. 6c 1373 Cells countet Cells with Nb% of cells with Nbs1GFP pos Nucleoli Laser+Mock Laser+Parpi 6 6 Mock+Mock Mock+parpi Cells countet Cells with Nb% of cells with Nbs1GFP pos Nucleoli Laser+Mock Laser+Parpi Mock+Mock Mock+parpi Cells countet Cells with Nb% of cells with Nbs1GFP pos Nucleoli Laser+Mock Laser+Parpi Mock+Mock Mock+parpi Supplementary Table 2 Statistical source data Table containing source data used for calculations of average, standard deviation and S.E.M. in experiments where n <