Fig. 2: Myosin VI protects stalled replication forks from degradation.

a Myosin VI is required for efficient unperturbed DNA replication. Top: Schematic representation of fiber assay conditions. Bottom: Fiber assays performed on siRNA-transfected U2OS cells. Left panel: replication speed, measured as total track lengths (CldU+IdU). Right panel: IdU/CldU ratios. b Depletion of myosin VI via siRNA causes erosion of stalled replication forks. Top: Schematic representation of fiber assay conditions. Bottom: Fiber assays performed on siRNA-transfected U2OS cells. c GFP-myosin VI complements the loss of endogenous myosin VI. U2OS cells harboring DOX-inducible GFP-myosin VI were siRNA-transfected (siRNA #10 targeting myosin VI) and treated −/+ 20 ng/ml DOX for 24 h, followed by fiber assays as shown in (b). d Myosin VI-dependent fork protection requires fork reversal factors. U2OS cells were siRNA-transfected as indicated, followed by fiber assays as shown in (b). e Motor- and MyUb-domains of myosin VI are required for fork protection. GFP-tail wildtype (WT) and mutants were overexpressed in U2OS cells, followed by fiber assays as shown in (b). Combined data from at least three independent replicates are shown. Detailed information about the respective mutations is given in Supplementary Fig. 2f. For all panels: IdU/CldU ratios are shown as dot plots with median values (red bars). Significance levels were calculated using the two-sided Mann–Whitney test from the indicated number of fibers per sample (ns: not significant, ****: p < 0.0001, ***: p < 0.001, **:p < 0.01, *: p < 0.05) and annotated for p > 0.0001. Knockdown efficiencies and overexpression levels are shown in Supplementary Fig. 2. For (a–d): A representative experiment from three independent replicates is shown. Source data are provided as a Source Data file.