Journal: bioRxiv

Article Title: An ATM-PPM1D Circuit Controls the Processing and Restart of DNA Replication Forks

doi: 10.1101/2025.05.13.652823

Figure Lengend Snippet: PPM1D regulates RAD51-mediated replication fork restart. (a) RAD51 foci analysis in U2OS cells. Top: experimental scheme. Bottom: dot plot shows the number of RAD51 foci in PCNA-positive cells pooled from three independent biological replicates. The mean number of RAD51 foci from each replicate is indicated by black dots. p -value was calculated using Student’s t-test. Error bars represent standard deviation. (b) Representative immunofluorescence images corresponding to (a). Scale bar, 10LJμm. (c) Immunoblot analysis of PPM1D, RAD51, and histone H4 in chromatin fractions from U2OS cells treated with or without PPM1Di. Ponceau S staining was used as a loading and total protein normalization control. (d) DNA fiber assay in U2OS cells. Top: experimental scheme showing treatment with PPM1Di and RAD51 inhibitor (RAD51i; B02). Bottom: bar graph shows the percentage of stalled replication forks under each condition. A minimum of 2959 fibers per condition were quantified from three independent biological replicates. p -values were calculated using one-way ANOVA. Error bars represent standard deviation. (e) RAD51 foci analysis in U2OS wild-type (WT) and PPM1D-knockout (KO) cells. Top: experimental scheme. Bottom: dot plot shows the number of RAD51 foci in PCNA-positive cells pooled from three independent biological replicates. The mean number of RAD51 foci from each replicate is indicated by black dots. p -values were calculated using one-way ANOVA with Tukey’s multiple comparisons test. Error bars represent standard deviation. (f) 53BP1 foci analysis in U2OS cells. Top: experimental scheme. Bottom: dot plot shows the number of 53BP1 foci in PCNA-positive cells pooled from three independent biological replicates. The mean number of 53BP1 foci from each replicate is indicated by black dots. p -values were calculated using one-way ANOVA with Tukey’s multiple comparisons test. Error bars represent standard deviation. (g) RAD51 foci analysis in U2OS WT and 53BP1-knockout (KO) cells. Top: experimental scheme. Bottom: dot plot shows the number of RAD51 foci in PCNA-positive cells pooled from three independent biological replicates. The mean number of RAD51 foci from each replicate is indicated by black dots. p -values were calculated using one-way ANOVA with Tukey’s multiple comparisons test. Error bars represent standard deviation.

Article Snippet: Primary antibodies include: β-actin (Proteintech, #66009-1-lg), β-tubulin (Proteintech, #66240-1-lg), Histone H4 (Bethyl Laboratories, #A300-647A), KAP1-pS824 (Bethyl Laboratories, #304-146A), Gamma-H2AX (Bethyl Laboratories, #A300-081A), RPA32 (Bethyl Laboratories, #A300-244A), PCNA (Santa Cruz Biotechnology, #sc-56), CHK1-pS345 (133D3, Cell Signaling Technology, #2348), PPM1D/WIP1 (Santa Cruz Biotechnology, #sc-376257), ATM-pS1981 (Cell Signaling Technology, #4526), 53BP1 (Cell Signaling Technology #4937), RAD51 (Millipore Sigma, #PC130), Rat anti-BrdU (BU1/75 (ICR1) (Abcam, #6326), Mouse anti-BrdU (B44) (BD Biosciences, #347580).

Techniques: Standard Deviation, Immunofluorescence, Western Blot, Staining, Control, Knock-Out