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SUPPLEMENTARY INFORMATION Supplementary Figure 1 FANCM controls DNA chain elongation. (A) FANCM was knocked down in the fibroblast cell line MRC5 (shFANCM plasmid used was 1). (B) MRC5 cells transfected with the empty vector or FANCM shRNA were pulse labeled with BrdU for 15 minutes. The median of replication fork track length is increased in the absence of FANCM. (C) The median of track length decreases in the absence of FANCM, when MRC5 cells were pulse-labeled for 60 minutes. The p-values were determined by Mann-Whitney testing. Supplementary Figure 2 FANCM promotes constant replication fork progression. (A) Control and FANCM shRNA expressing MRC5 cells were labeled with two consecutive pulses of 25 minutes. The track length of the first pulse (CldU) was plotted on the x-axis, while the corresponding length of the second pulse was represented on the y-axis. The linear regression shows a flatter angle when FANCM is depleted (right part of panel). The correlation coefficient r drops considerably in FANCM shRNA cells. (n > 50 for each condition). (B) Same experiment as in (A), but this time the DNA of MRC5 cells was pulse-labeled twice for 50 minutes. Supplementary Figure 3 FANCM opposes replication fork movement in the presence of hydroxyurea. (A) HeLa cells were exposed to different concentrations of hydroxyurea (HU) for 60 minutes in the presence of BrdU. DNA track length of more than 100 tracks was plotted on the y-axis for each concentration. DNA chain elongation was increased significantly in shFANCM (1) in the presence of HU. The p-values above the graph were generated by Mann-Whitney testing. (B) Cells were either transfected with pSUPER-puro (control) or FANCM shRNA containing plasmid and exposed for 3 hours to 10mM HU. In cells depleted for FANCM phosphorylation of Chk1 and H2AX are diminished. The reduction of monoubiquitination of FANCD2 in the FANCM shRNA cells confirmed that the knockdown was efficient. (C) FANCM shRNA cells survived better than control cells in the presence of increasing concentrations of HU, as determined by a cell survival assay. 1 Supplementary Figure 4 Cell cycle profiles and apoptotic activity of control and FANCM knockdown cells during treatment with hydroxyurea. (A) Representative cell cycle profiles of control and shFANCM cells treated for 0, 3, 6, and 24 hours with 2 mM hydroxyurea. Cells were fixed with ethanol, stained with propidium iodide, sorted by FACS (FacsScan, Becton Dickinson), and cell cycle profiles were analysed using FlowJo 8.8.6. Doublets and debris were eliminated by standard gating. (B) At the indicated time points, cells were lysed directly in 1x sample buffer, cell extracts were resolved by SDS PAGE, blotted onto nitrocellulose membrane and probed with anti-FANCM, anti-PARP1 and anti-actin antibodies. During apoptosis, PARP-1 (116 kDa) is cleaved by caspase-3 and-7 to yield 86 kDa and 27 kDa fragments. Supplementary Figure 5 Cell cycle profiles and apoptotic activity of control and FANCM knockdown cells during treatment with camptothecin. (A) Representative cell cycle profiles of control and shFANCM cells treated for 0, 3, 6, and 24 hours with 25 nM CPT. Cell cycle profiles were obtained as described in supplementary Figure 5. (B) At the indicated time points, apoptotic activity was evaluated by western blotting and probing with anti-PARP1 antibody to monitor apoptosis-induced cleavage of PARP1, as described in supplementary Figure 5. Supplementary Figure 6 FANCM promotes progression through UV-damaged DNA. (A) DNA was damaged by UV-C light and cells were instantly pulse labeled for 60 minutes with BrdU. Exposure to UV-C light decreased the BrdU track length in general. The median of track length in FANCM shRNA cells dropped by 40 % after exposure to 25J/m2 and by 29% after 50J/m2 relative to control cells treated with the same dose of UV light. P-values above the graph were generated by Mann-Whitney testing. (B) DNA damage checkpoint signaling was assessed by Western Blotting. HeLa cells were released after exposure to 50 J/m2 of UV light for up to three hours. FANCM shRNA cells showed diminished phosphorylation of Chk1 and H2AX. 2 Supplementary Figure 7 Cell cycle profiles and apoptotic activity of control and FANCM knockdown cells during treatment with UCN01. (A) Representative cell cycle profiles of control and shFANCM cells treated for 0, 3, 6, 12, and 24 hours with 100 nM UCN01. Cell cycle profiles were obtained as described in supplementary Figure 5. (B) At the indicated time points, apoptotic activity was evaluated by western blotting and probing with anti-PARP1 antibody to monitor apoptosis-induced cleavage of PARP1, as described in supplementary Figure 5. 3