Fig. 5: MCM8/9 function to direct SMARCAL1-mediated fork reversal and do not actively participate in stalled fork restart.
293T WT (gray), 8KO (blue), or 9KO (red) cells were transfected with siRNA twice for 24 h each to knockdown a SMARCAL1 (>150 fibers) or b HLTF (>100 fibers) and verified by western blot (*indicates a nonspecific band on the HLTF blot). Cells were then sequentially incubated with CldU and IdU for the indicated time intervals (○) or followed by 2 mM HU for 4 h (●). DNA was spread by gravity to measure NSD by quantifying fiber lengths using Image J. c Cells were treated with 2 mM HU and 10 µM mirin for 4 h to monitor fork restart as flanked by CldU and IdU labeling for indicated time intervals (>150 fibers). Cells were transfected with GFP-MCM8 (green outline, 
), GFP-MCM9 (green outline, 
), or GFP alone (Ø) (solid green, 
) as indicated for 24 h before initiating fork stalling and restart. d Cells were transfected with siRNA twice for 24 h each to knockdown MUS81 and then treated as in (a) and (b) (>140 fibers). IdU and CldU lengths were measured using ImageJ and the corresponding median ratios reported at the top of the plots and by a black line embedded in the data. Representative fibers are shown to the right of the dot plots. A Mann–Whitney two-sided U test was used to calculate P values (n.s. nonsignificant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).
