Fig. 2. Nuclear F-actin interaction with replication factories and nascent DNA.

a Representative image of a cell treated for 1 h with 100 nM CPT, stained for EdU and nuclear F-Actin (FLAG-NLS-Actin). Zoomed detail highlights EdU foci overlapping with F-Actin foci (yellow) quantified in Imaris. Scale bar = 10 μm b Quantification of Fig. 2a (see “Methods” for details) performed on 5 cells displayed as black dots. Box extends from the 25th to the 75th percentile. Line in the box represents the median. Whiskers extend down to the smallest and up to the highest value. c–f Isolation of proteins on nascent DNA (iPOND). c Top: Simplified experimental setup: 10 min EdU pulse in HEK293T cells optionally followed by a 50 min thymidine chase (Thy), discriminating chromatin-associated proteins behind replication forks. See Supplementary Fig. 2a for details. Bottom: Western blot analysis of cells optionally treated with CPT (100 nM, 1 h). Where indicated, LatB (100 nM) was added 10 min prior to CPT and retained. Proteins associated with nascent DNA were isolated by iPOND and detected with the indicated antibodies. Click reaction is performed using DMSO instead of biotin azide as a specificity control (Ctrl). d Graph-bar depicts mean ± SD of Actin protein levels at nascent DNA from three independent experiments (black dots). Values are normalized to H3 and displayed as fold change over NT. Statistical analysis: One-tailed t-test with Welch’s correction. e Western blot analysis of cells optionally treated with CPT (100 nM, 1 h). Where indicated, 100 nM CK666 was added 10 min prior to CPT and retained. Proteins associated with nascent DNA were isolated by iPOND and detected with the indicated antibodies. Ctrl sample as in (c). f Graph-bar depicts mean ± SD of Actin protein levels at nascent DNA from three independent experiments (black dots). Values are normalized to H3 and displayed as fold change over CPT. Statistical analysis: one-tailed t-test with Welch’s correction. Source data are provided as a Source Data file.