Figure 7.
ATRIP deficiency leads to increased spontaneous DNA damage, chromosomal sensitivity, and impaired survival upon exposure to genotoxic inducers requiring ATR pathway activation. (a) Immunofluorescence analysis of γH2AX foci in untreated EdU− fibroblasts from a HC, ATRIP patient (F1Pt), and ATR patient (F02-98). γH2AX nuclear foci were quantified and are shown in a dot plot representing pooled data from three independent experiments. At least 150 cells were analyzed for each condition. The median number of foci is depicted. ****P < 0.0001 (Kruskall–Wallis test and Dunn’s multiple comparisons test). (b) Schematic of principle and treatment protocols of the MN assays depicted in c and d. Cytokinesis is blocked with cytochalasin B (CytoB), allowing scoring of MN in BN cells. (c and d) Using the G0 and S MN assay, micronuclei were scored in untreated PHA blasts (c) and in PHA blasts exposed to 1 Gy of IR (d). The MMC MN assay was used to quantify micronuclei in PHA blasts treated with 0.02 µg/ml MMC (d). Mean MN values of a HC group are indicated by dashed lines. Dotted lines correspond to the mean + 3 SD and serve as a cut-off for sensitivity. Data of two independent experiments are shown for F1Pt, in each experiment an internal control (IC) was included. One experiment is performed for the family members (F1Si, F1Fa, F1Mo). (e) Bar graph depicts the total percentage of cell death after 120 h of culture for untreated fibroblasts of HC, F1Pt, and F02-98. Mean and SD of at least three independent experiments is shown. (f) Schematic outlining of the treatment protocol of the cytotoxicity assay used in g and h. (g and h) Fibroblasts from HC, F1Pt, and F02-98 were untreated or exposed to genotoxic inducers (0.2 µg/ml MMC, 20 J/m2 UV, or 10 Gy IR) without (g) and with (h) kinase inhibitors (20 nM ATRi or 10 µM ATMi) as indicated. Cell death was monitored by live imaging up to 120 h by quantifying the number of cells stained with SYTOX Green. Cell death kinetics are reflective of at least two independent experiments. (i) Bar graph indicating % cell death after 120 h of treatment for the experiments shown in g and h. Refer to the image caption for details.

ATRIP deficiency leads to increased spontaneous DNA damage, chromosomal sensitivity, and impaired survival upon exposure to genotoxic inducers requiring ATR pathway activation. (a) Immunofluorescence analysis of γH2AX foci in untreated EdU− fibroblasts from a HC, ATRIP patient (F1Pt), and ATR patient (F02-98). γH2AX nuclear foci were quantified and are shown in a dot plot representing pooled data from three independent experiments. At least 150 cells were analyzed for each condition. The median number of foci is depicted. ****P < 0.0001 (Kruskall–Wallis test and Dunn’s multiple comparisons test). (b) Schematic of principle and treatment protocols of the MN assays depicted in c and d. Cytokinesis is blocked with cytochalasin B (CytoB), allowing scoring of MN in BN cells. (c and d) Using the G0 and S MN assay, micronuclei were scored in untreated PHA blasts (c) and in PHA blasts exposed to 1 Gy of IR (d). The MMC MN assay was used to quantify micronuclei in PHA blasts treated with 0.02 µg/ml MMC (d). Mean MN values of a HC group are indicated by dashed lines. Dotted lines correspond to the mean + 3 SD and serve as a cut-off for sensitivity. Data of two independent experiments are shown for F1Pt, in each experiment an internal control (IC) was included. One experiment is performed for the family members (F1Si, F1Fa, F1Mo). (e) Bar graph depicts the total percentage of cell death after 120 h of culture for untreated fibroblasts of HC, F1Pt, and F02-98. Mean and SD of at least three independent experiments is shown. (f) Schematic outlining of the treatment protocol of the cytotoxicity assay used in g and h. (g and h) Fibroblasts from HC, F1Pt, and F02-98 were untreated or exposed to genotoxic inducers (0.2 µg/ml MMC, 20 J/m2 UV, or 10 Gy IR) without (g) and with (h) kinase inhibitors (20 nM ATRi or 10 µM ATMi) as indicated. Cell death was monitored by live imaging up to 120 h by quantifying the number of cells stained with SYTOX Green. Cell death kinetics are reflective of at least two independent experiments. (i) Bar graph indicating % cell death after 120 h of treatment for the experiments shown in g and h.

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