Supplementary Figure 1

Transcription

1 Supplementary Figure 1 Cell cycle distribution (%) Cell Cycle G1 S G2 Viability (%) Viability Supplementary Figure 1. Cell cycle distribution and viability during DSB repair measurements. and were cultivated and nucleofected with a DNA mixture consisting of pcmv-i-scei, homologous repair substrate HR-EGFP/3 EGFP and pbs as in Figure 2. Following recultivation for 24h in medium the percentages of viable cells with DNA content in G1, S, or G2 cell cycle phases were determined with propidium iodide stained cells. Viability of cells indicated by a DNA content higher than sub-g1 was plotted. Columns display mean values from 3-5 and samples, bars, SEM.

2 Supplementary Figure 2 MLLbcr control 12 1 Band intensity (%) h 9h 1h 2h 6h 9h untreated Supplementary Figure 2. Radiation-induced breakage of the MLL breakpoint cluster region (MLLbcr). and were cultivated for 72h followed by further cultivation (untreated) or irradiation with a dose of 2Gy of photons () and recultivation for the indicated times. Genomic PCR analysis showed reduced MLLbcrspecific amplification (band intensity) indicating breakage 2h post- in and. The image on top shows a representative agarose gel with the electrophoresed PCR products, the diagram below displays mean band intensities ±SEM from 4 independent experiments, whereby MLLbcr-specific band intensities were individually normalized to a control sequence in MLL intron 2, and resulting values for untreated, 9h, set to 1% each.

3 Supplementary Figure 3 DSB repair frequency (%) 15 Caffeine 1 5 Supplementary Figure 3. Homologous repair in response to Caffeine treatment. and were cultivated and nucleofected with a DNA mixture consisting of pcmv-i-scei, homologous repair substrate EGFP/3 EGFP and pbs or wild-type EGFP expression plasmid as in Figure 2. Following recultivation for 24h in medium without or with 2mM Caffeine cells were FACS analyzed for EGFP-positivity. Mean values for mock-treated samples were set to 1% for each cell type and experimental day. Columns, mean values, bars (n=3-9), SEM;

4 Supplementary Figure Disulfiram (µm) Supplementary Figure 4. Effect of NF- B inhibition on spontaneous 53BP1 foci formation. and were DMSO-treated or treated with the NF- B inhibitor Disulfiram at the indicated concentrations (.2, 2, 4 M). After an incubation period of 5h the cells were fixed and 53BP1 foci assembly was detected by immunofluorescence microscopy. Foci were scored by automated quantification from 3-4 independent experiments with 3 nuclei per experiment, foci/cell values normalized to the DMSO control and statistical significance of differences calculated as for Figure 5 between drug and DMSO-treated cells using nonparametric Mann-Whitney test for unpaired samples; P<.1.

5 Supplementary Figure 5 Cell cycle distribution (%) Cell Cycle G1 S G Disulfiram Supplementary Figure 5. Cell cycle distribution after Disulfiram treatment. and were cultivated and treated with and without 4 M Disulfiram and the percentages of viable cells with DNA content in G1, S, or G2 cell cycle phases were determined with propidium iodide stained cells. Columns display mean values from 3-5 and samples, bars, SEM.

6 Supplementary Figure 6 (a) (b) (c) Caffeine DNA-PKi Caffeine + DNA-PKi (d) 15 IQD (e) 15 Caffeine + DNA-PKi + IQD (f) 15 Trichostatin A (g) 15 Aza-dC BP1 relátive foci (%) 1 5 Supplementary Figure 6. Focal 53BP1 accumulation as a function of distinct damage signaling steps. and were DMSO-treated or pretreated with the indicated pharmacological inhibitors for 2-4h and exposed to 2Gy -ray (). After an incubation period of 1h the cells were fixed and 53BP1 foci assembly was detected by immunofluorescence microscopy. Foci were scored by automated quantification in 1 nuclei per experiment in 2-6 independent experiments, foci/cell values normalized to the irradiated DMSO control and statistical significance of differences calculated as for Figure 5 using nonparametric Mann-Whitney test for unpaired samples ; P<.5, P<.1, P<.1, P<.1. (a) Caffeine treatment (2mM; C). (b) DNA-PKi (1µM NU7441; D). (c) Caffeine and DNA-PKi (NU7441) cotreatment (2mM and 1µM, respectively). (d) PARPi (75µM IQD; I). (e) Caffeine, DNA-PKi (NU7441) and PARPi (IQD) cotreatment (2mM, 1µM and 75µM, respectively). (f) Trichostatin A treatment (1.5µM; T). (g) Aza-dC treatment (1µM, A).

7 Supplementary Figure 7 EGFP positive cells [%] I B -SR Supplementary Figure 7. NF- B reporter assay after expression of superrepressor I B -SR. and were cultivated and nucleofected with a DNA mixture mimicking DSB repair assay conditions and including reporter plasmid for NF-kB-dependent GFP production as in Figure 6a. Afterwards cells were recultivated for 24h and the percentage of green fluorescent cells determined by flow cytometry. Columns show mean values (n=9-15), bars, SEM. Statistical significance of differences between controls ( versus ) and control versus I B -SR samples were determined using the nonparametric Mann-Whitney test for unpaired samples (GraphPad Prism 5.1). P<.1; P<.1.