Figure 6.
Loss of ATRIP results in compromised DNA replication and impaired cell cycle progression following replication stress. (a) Schematic outlining the treatment protocol of the FCM assay used in b and c. (b) Cell cycle distributions of untreated fibroblasts from a HC and ATRIP patient (F1Pt). Representative FCM plots are shown (left). Scatter dot plot depicts data from eight independent experiments (right). Mean and SD are shown. ns: not significant (multiple paired t tests). (c) FCM EdU pulse-labeling profiles of MMC treated HC and F1Pt fibroblasts (left). Cells were exposed to 0.02 µg/ml MMC for 24 h in the absence or presence of an ATR kinase inhibitor (ATRi, 20 nM). Histograms of EdU and DAPI intensity in S phase cells are shown (right). The median fluorescence intensity (MFI) is annotated on the histogram. Data are representative of three independent experiments. (d) Quantification of RPA nuclear fluorescence in HC and F1Pt fibroblasts 3 h after 1 mM HU exposure and concomitant EdU pulse-labeling. The mean RPA intensity per nucleus is shown for EdU+ cells. Dot plot represents data from pooled data from three experiments. The median value is depicted. ns: not significant; ****P < 0.0001 (multiple Mann–Whitney tests and Bonferroni–Dunn multiple comparisons test). (e) FCM EdU pulse-labeling profiles of HC and F1Pt fibroblasts after the release from HU treatment (left). Cells were exposed to 1 mM HU for 3 h, released for 3 h, and subsequently harvested. Histograms of EdU intensity in S phase cells are shown (right). Data are representative of two experiments. (f) Cell cycle profiles of HC and F1Pt fibroblasts following 72 h of 0.02 µg/ml MMC treatment, with and without 20 nM ATRi. The percentage of cells in G2/M phase is indicated. Data are representative of three independent experiments. (g) EdU pulse-chase kinetics of HC and F1Pt PHA blasts. Cells were untreated or treated with genotoxic inducers (0.02 µg/ml MMC or 200 J/m2 UV), pulse-labeled with EdU, and harvested at indicated time points. Kinetic plots show percentages of EdU+ cells present in S phase and are representative of three independent experiments. (h) CTV profiles of CD8+ and CD4+ PHA blasts from HC and F1Pt after 96 h of culture in the presence or absence of 0.02 µg/ml MMC. Data are representative of two experiments. Refer to the image caption for details.

Loss of ATRIP results in compromised DNA replication and impaired cell cycle progression following replication stress. (a) Schematic outlining the treatment protocol of the FCM assay used in b and c. (b) Cell cycle distributions of untreated fibroblasts from a HC and ATRIP patient (F1Pt). Representative FCM plots are shown (left). Scatter dot plot depicts data from eight independent experiments (right). Mean and SD are shown. ns: not significant (multiple paired t tests). (c) FCM EdU pulse-labeling profiles of MMC treated HC and F1Pt fibroblasts (left). Cells were exposed to 0.02 µg/ml MMC for 24 h in the absence or presence of an ATR kinase inhibitor (ATRi, 20 nM). Histograms of EdU and DAPI intensity in S phase cells are shown (right). The median fluorescence intensity (MFI) is annotated on the histogram. Data are representative of three independent experiments. (d) Quantification of RPA nuclear fluorescence in HC and F1Pt fibroblasts 3 h after 1 mM HU exposure and concomitant EdU pulse-labeling. The mean RPA intensity per nucleus is shown for EdU+ cells. Dot plot represents data from pooled data from three experiments. The median value is depicted. ns: not significant; ****P < 0.0001 (multiple Mann–Whitney tests and Bonferroni–Dunn multiple comparisons test). (e) FCM EdU pulse-labeling profiles of HC and F1Pt fibroblasts after the release from HU treatment (left). Cells were exposed to 1 mM HU for 3 h, released for 3 h, and subsequently harvested. Histograms of EdU intensity in S phase cells are shown (right). Data are representative of two experiments. (f) Cell cycle profiles of HC and F1Pt fibroblasts following 72 h of 0.02 µg/ml MMC treatment, with and without 20 nM ATRi. The percentage of cells in G2/M phase is indicated. Data are representative of three independent experiments. (g) EdU pulse-chase kinetics of HC and F1Pt PHA blasts. Cells were untreated or treated with genotoxic inducers (0.02 µg/ml MMC or 200 J/m2 UV), pulse-labeled with EdU, and harvested at indicated time points. Kinetic plots show percentages of EdU+ cells present in S phase and are representative of three independent experiments. (h) CTV profiles of CD8+ and CD4+ PHA blasts from HC and F1Pt after 96 h of culture in the presence or absence of 0.02 µg/ml MMC. Data are representative of two experiments.

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