Transcription

1 DOI: /ncb3259 A Ismail et al. Supplementary Figure 1 B SSC Live cells FSC- PARR PARR Width FSC Single cells FSC- PARR GFP-RFP+ cells RFP 10 2 C 10 1 GFP+RFP- cells GFP 10 4 GFP+RFP- cells 10 3 RFP GFP 10 4 GFP-RFP+ cells 10 3 RFP GFP Supplementary Figure 1 The RNF138 amino acid sequence is conserved. (A) Amino acid alignment of RNF138 from different organisms is shown. Alignments was performed using Uniprot online tools. Identical residues are highlighted in black, conserved hydrophobic resides in light grey, other conservative changes are shown in darker grey. Star symbols indicate the conserved residues. (B) Scatter plots of the flow cytometric analysis of HeLa-Fucci cells prior to and post-sorting. The target cell populations were sorted using the indicated gates. (C) Western blot analysis of extracts prepared from sorted cells. Whole-cell extracts were prepared from sorted Fucci cells and probed for Ku80, cyclin A (S/G2 marker), and tubulin (loading control) by immunoblotting. 1

2 A B C Supplementary Figure 2 RNF138 knock down alters Ku80 retention at the sites of DNA damage. (A) GFP-Ku80 expressing cells were either transfected with control sirna (CTL sirna) or one of two different RNF138 sirna (RNF138 sirna1 and RNF138 sirna2) for 48 hr. Recruitment of GFP-Ku80 to sites of laser induced DNA damage after micro irradiation in U2OS cells was monitored using time lapse microscopy. Representative images are shown. Right: The mean levels of Ku80 accumulation at sites of laser tracks was quantified using LSM software and plotted as indicated. Data show mean +/- s.d; n=44 cells, data pooled across two independent experiments. (B) RNF138 does not target Ku80 for degradation. U2OS cells transfected with either control sirna (CTL sirna) or RNF138 sirna were treated with cycloheximide (CHX, 40 μm alone or together with MG132 (MG132, 20 μm) for indicated periods. The degradation of endogenous Ku80 was analyzed by anti-ku80 immunoblotting. (C) Efficiency of NHEJ repair in NHEJ reporter cells reconstituted with different sirna resistant RNF138 deletion mutant constructs and transfected with control sirna (CTL sirna) or RNF138-specific sirna (RNF138 sirna). The percentage of GFP positive cells was measured using a flow cytometer. Data show the mean +/- SD; n=3 biologically independent experiments that were based on gating 20,000 in each experimental condition. Scale bars are 5 μm. 2

3 Supplementary Figure 3 RNF138 mediates Ku80 ubiquitylation in response to DNA damage in S/G2 phase of the cell cycle. Fucci cells were either transfected with control sirna (CTL sirna) or RNF138 specific sirna (RNF138 sirna) and then sorted by flow cytometry. Cells were exposed or not exposed to DNA damage by CLM. Soluble (Sol.) and chromatin fraction (Chr.) were isolated and subjected to immunopreciptation as indicated. Images are representative of two independent experiments. 3

4 Supplementary Figure 4 Cell cycle synchronization of DR95 cells expressing Isce-I cutting site. Cells were synchronized, DNA content analysis was done by flow cytometer using propidium iodide staining. Left: Representatives DNA content profiles were shown. Right: the percentage of cells at each phase of the cell cycle is represented in the form of a table. Images are representative of two independent experiments. 4

5 Ku80 NBS1 Merge No CPT CPT CPT+ATMi CPT+ RNF138 sirna1 CPT+ RNF138 sirna2 CPT+Mirin 200 Ku80 foci/cell No CPT CPT CPT+ATMi CPT+ Mirin CPT+ RNF138 sirna2 CPT + RNF138 sirna1 Supplementary Figure 5 RNF138 knock down inhibits Ku removal from the sites of DNA damage in S phase. (a) Cells were treated with CPT (1 μm, for 1 hr) in the presence or absence of different treatments: ATM inhibitor (10 μm, added 1 hr before CPT treatment, Mirin (100 μm, added 1 hr before CPT treatment) or transfected with two different RNF138 sirna (RNF138 sirna1 and RNF138 sirna2) for 48 hrs before CPT (1 μm, for 1 hr). Images were captured using Z-stacks, deconvolved using Hygens deconvolution software, Ku80 foci were quantified using MetaXpress Software and plotted on the bottom. Average Ku80 foci per cell were counted and plotted as indicated. Data show mean +/- s.d; n=42 cells that were obtained from two biological replicates. Scale bars are 10 μm. 5

6 A YFP-Mre11 B C YFP-Mre11 on laser stripe RNF138 sirna1 RNF138 sirna2 CTL sirna sec 582 sec Supplementary Figure 6 Effect of RNF138 depletion on the recruitment of Mre11 to sites of DNA damage. (A) U2OS cells were co-transfected with the indicated sirnas and YFP-Mre11 plasmids for 48 hours. Cells were then laser-microirradiated and the recruitment of YFP-Mre11 to the sites of DNA damage was monitored by time-lapse microscopy. Signal intensity of YFP Mre11 at DNA damage sites relative to the unirradiated area was quantified. Representative pictures are shown. (B) Western blot analysis of cells to estimate the knock down efficiency of RNF138 are shown and served as a control. (C) The mean levels of YFP-Mre11 accumulation at sites of laser tracks was quantified using LSM software and plotted as indicated. Data show mean +/- s.d; n=34 cells, data pooled across two biologically independent experiments. Scale bars are 10 μm. 6

7 A Time (sec) t=0 t=12 t=300 Ismail et al. Supplementary Figure 7 DMSO GFP-RNF138 Mirin CTL sirna B Ku80 sirna Mre11 sirna C Supplementary Figure 7 RNF138 acts downstream of Mre11 activity in the HR pathway. (A) U2OS cells were co-transfected with the indicated sirnas and GFP-RNF138 for 48 hours. In the case of Mirin treatment, cells transfected with GFP-RNF138 were pre-treated with Mirin (100 μm, added 1 hr before laser microirradiation). Cells were then laser-microirradiated and the recruitment of GFP-RNF138 to the sites of DNA damage was monitored by time-lapse microscopy. Representative pictures were shown on the left. Quantification of GFP-RNF138 signal on laser- induced DNA damage stripes were shown on the right. Data show mean +/- s.d; n=38 cells, data pooled across two biologically independent experiments. (B) Western blot analysis of cells to estimate the knock down efficiency of Mre11 and Ku80 are shown and served as a control. (C) Cell cycle profiles of RNF138 depleted cells. U2OS cells were transfected with control sirna (CTL sirna) or one of two different RNF138 sirna (RNF138 sirna1 and RNF138 sirna2) for 48 hours. Cells were harvested and subjected to DNA content analysis using propidium iodide staining according to standard protocols. Cell cycle graphs are shown on the left. A table summarizing the cell cycle results are shown on the right. At least cells were analyzed. Scale bars are 5 μm. 7

8 A MDC1 53BP1 DAPI CTL sirna RNF138 sirna1 RNF138 sirna2 100 B MDC1 IRIF -positive cells with 53BP1 IRIF CTL sirna RNF138 sirna1 RNF138 sirna2 kda Supplementary Figure 8 RNF138 did not appreciably alter MDC1/53BP1 IRIF. Immunostaining of U2OS cells transfected with either control (CTL) sirna or one of two different RNF138 sirnas (RNF138 sirna1 and RNF138 sirna2) for 48 hrs. Cells were then exposed to 2 Gy and allowed to recover for 1 hr before being fixed and immunostained as indicated. Bottom: quantification of populations of cells with >15 MDC1 foci colocalized with 53BP1 were plotted by counting 100 cells per experiment. Data show mean +/- s.d; n=200 cells, data pooled across two biologically independent experiments. (B) U2OS cells were co-transfected with either control (CTL sirna) or one of two different RNF138 sirna (RNF138 sirna1 and RNF138 sirna2) targeting RNF138 and sirna resistant GFP-RNF138 for 48 hrs. Cells were harvested and nuclear extracts were prepared and immunoblotted as indicated. Scale bars are 5 μm. 8

9 Full scans of immunoblots Fig. 2b Fig. 2e Fig. 2f Fig. 3b Fig. 3c Fig. 3d Fig. 3e Fig. 3f Supplementary figure 9 Full gel scans 9

10 Full scans of immunoblots Fig. 3h Fig. 3g Sup. Fig. 7b Sup. Fig. 3 Supplementary figure 9 continued 10

11 Full scans of immunoblots Fig. 5 Fig. 7a Suppl. Fig. 2b Suppl Fig. 3 Fig. 8e Supplementary figure 9 continued 11

12 Supplementary Table Legends Supplementary Table 1 Summary of the E3 ubiquitin ligases library used in the study and their corresponding z-scores. Supplementary Table 2 A list of RNF138 interacting proteins identified by mass spectrometry. For details see supplementary experimental procedures. Supplementary Table 3 A list of antibodies used in the study. Supplementary Table 4 A list of PCR primers used in the study. Supplementary Table 5 A list of sirna sequences used in the study. Supplementary Table 6 A list of DNA probe sequences used in the EMSA experiments. 12