GEN MUS8 GEN MUS8 GEN MUS8 GEN MUS8 GEN C LM MUS8 XPF (loading control) D H2AX Frequency of -positive bridges (% of anaphase cells) 6 4 2 p =.8 x -4 GM855 p =.27 PSNF5 E H2AX Figure S. Analysis of anaphase bridges and lagging chromosomes in - depleted HeLa cells. (A) HeLa cells were treated with the indicated s and the efficiency of protein depletion was analyzed in whole cell extracts by western blotting. Since co-depletes with, levels were used to monitor depletion. () Quantification of -positive anaphase bridges in control or -treated, GM855 (LM-deficient) and PSNF5 (GM855- derived, LM-complemented) cells. 335 anaphase cells were analyzed; p values were determined using a two-tailed Fisher s exact test. (C) Localization of LM (red) on -positive anaphase bridges and segregating daughter DNA masses. (D) γh2ax-staining (red) on visible gaps on a -positive anaphase bridge. (E) Exteive γh2ax coating on a -positive bridge in a late anaphase/telophase cell. All representative images were obtained from cisplatintreated, -depleted HeLa cells. Scale bar, µm. Arrows indicate points of interest.
si p <. si si SCEs per chromosomes per metaphase 5 5 p <. Figure S2. and -depleted cells show reduced levels of cisplatininduced SCE-formation. (A) Representative images of metaphase spreads prepared from control, and -depleted HeLa cells following treatment with cisplatin. Scale bar, µm. () Quantification of cisplatin-induced SCEs in control, and -depleted HeLa cells. Each datapoint represents a single metaphase; in total, 46 metaphases and >2,8 chromosomes were screened for SCEs. lack bars represent the mean number of SCEs per chromosomes per metaphase spread. p values were determined using a two-tailed t-test, = not significant
53P p =.5 p = 2.56 x -8 p = 2.63 x -6 cyclina MERGE Cyclin A-positive cells containing 53P foci (%) 5 4 3 2 2% 33% MUS8 GEN 37% GEN 22% GEN si sigen Figure S3. Cyclin A-positive HeLa cells depleted for SLX-MUS and GEN pathway protei exhibit elevated levels of 53P foci. (A) Representative images of 53P foci (purple) in cyclin A-positive (green), GEN- depleted HeLa cells. Scale bar, 6 µm. () Quantification of 53P focusformation in cyclin A-expressing control, MUS8 GEN, GEN and GEN -treated HeLa cells (n = 72 cyclin A-positive cells). p values were determined using a two-tailed Fisher s exact test.
A si si & sigen p <. Segmented chromosomes per metaphase spread (%) 5 4 3 2 SL CO NT RO X L G EN SL X4 G M EN US 8 G EN si sigen C simus8 sigen D si (untreated) p <.5 Segmented chromosomes per metaphase spread (%) 5 4 3 2 cisplatin - - - si (cisplatin-treated) Figure S4., but not, depletion promotes chromosome segmentation in S cells. (A) Representative images of metaphase spreads prepared from GM855 S cells treated with the indicated s. Scale bar, µm. () Quantification of segmented chromosomes per metaphase spread in S cells treated with the indicated s (n = 7 metaphase spreads). lack bars represent the mean ± SD for each dataset. p values were determined using twotailed t-tests; = not significant. (C) Representative images of metaphase spreads prepared from untreated (top) and cisplatin-treated (bottom) depleted S cells. Scale bar, µm. (D) Quantification of segmented chromosomes per metaphase spread in S cells treated with the indicated s and with or without cisplatin (n = 56 metaphase spreads). The data was analyzed as indicated in ().
cell survival (%) MUS8 GEN MUS8 MUS8 GEN.5..5 2. Hydroxyurea (mm) G S G2 GEN MUS8 GEN MUS8 MUS8 GEN GEN MUS8 8 6 4 2 2 % DNA content distribution (PI) Mitotic fraction (ph3) Figure S5. DNA damage seitivity and DNA content distributio of resolvasedepleted cells. (A) Survival of HeLa cells treated with the indicated s, following exposure to increasing doses of hydroxyurea. Error bars, ± SEM. () DNA-content distributio (PI; left) and mitotic fractio (phospho-histone H3; right), following depletion of the indicated protei in asynchronous populatio of undamaged HeLa cells.
Figure S6. Analysis of cell-cycle progression in undamaged and damaged cells following resolvase depletion. (A) Cell-cycle progression of rdu-positive HeLa cells following treatment with control or GEN s and cisplatin. () Cell-cycle progression of control,, GEN and GEN-depleted rdu-positive HeLa cells. (C) Kinetics of cell-cycle progression of rdu-positive cells, as shown in ().
Supplementary Table A. 95% confidence intervals corresponding to the mean levels of micronuclei-formation (Fig. 3A) and multinucleation (Fig. 3D) observed in SLX-MUS and/or GEN-depleted HeLa cells. % Micronuclei- formation (95% confidence intervals) % Multinucleation (95% confidence intervals) Undamaged cells Damaged cells Undamaged cells Damaged cells.3 4.7.4 3.6.3 2..4 2.9 MUS8 2. 6..7 5.5.7 2.5.6 3.3 GEN 3. 7.5.3 5..2 2.4 2.6 6.8 MUS8 & GEN 5.3.9 2. 2.3 2.2 6.4 6.4 2.4 3.2 7.9 5.3 24..3 3. 5.7.4 & MUS8 4.3 9.3 2.8 2.4.5 3. 6.2.9 & GEN 5.3 23. 32.9 44. 3.9 8.7 9.2 27.5 3. 7.6 2.5 7.2.3.8 4. & MUS8 4.2 9. 5.8 2.3.6.8 5.5 & GEN 6..8.7 8.4.7 3.6.9 5.7. Comparison of levels of micronuclei-formation and multinucleation in cells treated with the indicated s. Significance was determined using a two-tailed Fisher s exact test. Comparison of micronuclei- levels (Fisher s exact 2- tailed p- values) Undamaged cells Damaged cells Comparison of multinucleation- levels (Fisher s exact 2- tailed p- values) Undamaged Damaged cells cells vs..42.8.689.583 vs..4.8 x -3.53.9 x -4 vs. MUS8.447.252.546 vs. GEN.3.462.752.44 MUS8 vs. MUS8 & GEN.22 2.2 x -8.35.78 x -5 GEN vs. MUS8 & GEN.47 9.39 x -9.3.92 vs. & MUS8.449.694.485 vs. & GEN.62 2.73 x -5.7.368 vs. & MUS8.547.466.887 vs. & GEN.2 x -8 4. x -7. 5.99 x -7