Journal: The EMBO Journal

Article Title: Spatial mapping of DNA synthesis reveals dynamics and geometry of human replication nanostructures

doi: 10.1038/s44318-025-00574-2

Figure Lengend Snippet: ( A ) Representative 2D maximum projections of 3D-SIM images of an early and mid S-phase nucleus after labelling with the standard pulsing scheme and post-fixation PCNA immunofluorescence staining, scale bar = 5 µm. ( B ) Bar plot showing the mean correlation coefficient (Pearson’s R value with no threshold) between indicated image channels, as determined by the FIJI plugin Coloc 2, n = 4. Error bars represent SD. ( C ) Examples of nuclear areas containing individual replication events, with arrows indicating inferred directionality, scale bar = 500 nm. For each area, an EdU only, Cy3-xx-dUTP only, PCNA only and merged channel image are shown below. ( D ) Bar plot showing the mean nuclear integrated intensities (a.u.) of pH2AX and pRPA for cells treated as described in 1E, F, respectively. Three replicates per condition, error bars represent SD. ( E ) Representative 2D maximum projections of 3D-SIM images of cells after three different treatments: EdU for 30-min only, EdU for 30-min and Cy3-xx-dUTP for 30 (4 + 26)-min or 2-h HU treatment prior to labelling with EdU for 30-min followed by Cy3-xx-dUTP for 30 (4 + 26)-min. All conditions underwent post-fixation immunofluorescence staining of pH2AX, scale bars: 10 µm. The nuclear perimeter is annotated with a white dashed line, and a selected area (white square) is shown in the merged image, scale bars: 2 µm. ( F ) Representative 2D maximum projections of 3D-SIM images of cells after three different treatments: EdU for 30-min only, EdU for 30-min and Cy3-xx-dUTP for 30 (4 + 26)-min or 2-h HU treatment prior to labelling with EdU for 30-min followed by Cy3-xx-dUTP for 30 (4 + 26)-min. All conditions underwent post-fixation immunofluorescence staining of pRPA, scale bars: 10 µm. The nuclear perimeter is annotated with a white dashed line, and a selected area (white square) is shown in the merged image, scale bars: 2 µm. .

Article Snippet: If immunostaining applied, fixed samples were incubated overnight at 4 °C with a primary antibody: phospho-histone H2A.X [Ser139] (1:800, Cell Signalling Technology, #2577); phospho-RPA32 [S33] (1:2000, Bethyl Laboratories, #A300-246A); or PCNA (PC10) (1:400, Santa Cruz, sc-56) in blocking media.

Techniques: Immunofluorescence, Staining

Journal: The EMBO Journal

Article Title: Spatial mapping of DNA synthesis reveals dynamics and geometry of human replication nanostructures

doi: 10.1038/s44318-025-00574-2

Figure Lengend Snippet: ( A ) Bar plot showing the mean nuclear integrated intensities (a.u.) of MCM2, PCNA and EdU for asynchronous cells treated with Doxycycline for 24 h and DMSO control. G1 and S population were selected based on DAPI vs EdU. Three replicates per condition, error bars represent S.E.M (for each condition, at least 10,000 cells were analysed for G1 portion and at least 2000 cells were analysed for the S portion).

Article Snippet: If immunostaining applied, fixed samples were incubated overnight at 4 °C with a primary antibody: phospho-histone H2A.X [Ser139] (1:800, Cell Signalling Technology, #2577); phospho-RPA32 [S33] (1:2000, Bethyl Laboratories, #A300-246A); or PCNA (PC10) (1:400, Santa Cruz, sc-56) in blocking media.

Techniques: Control