Extended Data Fig. 6: Validation of CRISPR-engineered 53BP1-PCNA RPE-1 cells.
From: Multigenerational cell tracking of DNA replication and heritable DNA damage
(a) 53BP1-PCNA RPE-1 cells were treated with siRNA for 72 h and IR (4 Gy, 2 h) as indicated and stained for 53BP1 (Cy5). The endogenous 53BP1-mScarlet and antibody-based signals are shown, together with their segmentation masks. Scale bar, 10 µm. (b) High-content microscopy-based quantification of 53BP1-mScarlet foci using the samples and foci segmentation mask from (a). Statistical analysis by one-way ANOVA followed by Tukey’s post hoc test. (c) Video stills of 53BP1-PCNA RPE-1 cells at different cell cycle phases from G1 through S-phase and to G2. PCNA-mEmerald patterns and their segmentation are shown. Scale bar, 10 µm. (d) DAPI- and EdU-based cell cycle gating of 53BP1-PCNA RPE-1 cells and PCNA foci analysis in different cell cycle phases (colour-coded based on DAPI and EdU). (e) PCNA foci quantification from (d) in G1 versus S-phase versus G2 (based on DAPI and EdU). Statistical analysis by one-way ANOVA followed by Tukey’s post hoc test. (f) Cell cycle resolved scatter plots of γΗ2ΑΧ mean intensities from untreated RPE-1 cells or RPE-1 cells treated with 0.2 µM aphidicolin or 1 µM ATR inhibitor for 48 h. (g) Cell cycle resolved scatter plots of pRb mean intensities from untreated RPE-1 cells or RPE-1 cells treated with 0.2 µM aphidicolin or 1 µM ATR inhibitor for 48 h. (h) Cell cycle resolved scatter plots of p53 mean intensities from untreated RPE-1 cells or RPE-1 cells treated with 0.2 µM aphidicolin or 1 µM ATR inhibitor for 48 h. (i) Cell cycle resolved scatter plots of p21 mean intensities from untreated RPE-1 cells or RPE-1 cells treated with 0.2 µM aphidicolin or 1 µM ATR inhibitor for 48 h. (j) Western blot analysis of phospho-KAP1 in untreated RPE-1 cells or RPE-1 cells treated with 0.2 μM APH or 1 μM ATRi for 24 h. For gel source data, see Supplementary Fig. 1.
