Figure S5.
Loss of ATRIP increases DNA damage, chromosomal instability, and reduced cellular fitness. (a) Representative immunofluorescence images with DAPI and γH2AX staining of data shown in Fig. 7 a. γH2AX foci are shown in untreated fibroblasts from a control (HC), ATRIP patient (F1Pt), and ATR patient (F02-98). Images are representative of three independent experiments. Scale bars are 20 µm. (b) Using the G0 and S MN assay, micronuclei were scored in PHA blasts exposed to 0.5 Gy IR. Mean MN values of a reference HC group are indicated by dashed lines. Dotted lines correspond to the mean of HCs + 3 SD and serve as a cut-off for sensitivity to IR. Data of two independent experiments are shown for F1Pt, in each experiment an internal control (IC) was included. One experiment was performed for the family members (F1Si, F1Fa, F1Mo). (c and d) Fibroblasts from HC, F1Pt, and F02-98 were exposed to genotoxic inducers (0.2 µg/ml MMC, 20 J/m2 UV, or 10 Gy IR) in combination with a specific kinase inhibitor (2 µM DNA-PKi) (c) or only treated with 20 nM ATRi, 10 µM ATMi, or 2 µM DNA-PKi (d). Cell death was monitored by live imaging up to 120 h by quantifying the number of cells stained with SYTOX Green. Represented cell death kinetic plots are reflective of at least two independent experiments. (e and f) Corresponding proliferation curves of the data shown in Fig. 7, 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 (e) or with (f) specific kinase inhibitors (20 nM ATRi or 10 µM ATMi) as indicated. Percentage of confluency was monitored by live imaging up to 120 h. Data are representative of at least two independent experiments. (g) Schematics of genome editing of the ATR and ATRIP allele (top). Blue lines represent the location of crRNAs, orange arrows depict the joint primers annealing to sequence outside the crRNA cutting regions, and red arrows represent the primers annealing to depletion region, used for wild type (WT) allele detection. The western blot verification of mono-allelic cells is shown (bottom). Cells were seeded in 6 cm dish, and 1 μg/ml doxycycline was added in the culture medium 24 h before cell lysis to induce the expression of cas9 protein. (h) Protein expression of ATR, ATRIP, RPA1, RPA2, and TOPBP1 in the novel generated MCF10A mono-allelic cells. β-Actin was used as loading control. (i) Cell fitness of MCF10A mono-allelic cells with frameshift mutations serving as internal control. Each variant has three independent biological replicates. Mean and SEM are shown. *P < 0.05, **P < 0.01, ***<0.001 (two-tailed paired t tests). Source data are available for this figure: SourceDataFS5. Refer to the image caption for details.

Loss of ATRIP increases DNA damage, chromosomal instability, and reduced cellular fitness . (a) Representative immunofluorescence images with DAPI and γH2AX staining of data shown in Fig. 7 a. γH2AX foci are shown in untreated fibroblasts from a control (HC), ATRIP patient (F1Pt), and ATR patient (F02-98). Images are representative of three independent experiments. Scale bars are 20 µm. (b) Using the G0 and S MN assay, micronuclei were scored in PHA blasts exposed to 0.5 Gy IR. Mean MN values of a reference HC group are indicated by dashed lines. Dotted lines correspond to the mean of HCs + 3 SD and serve as a cut-off for sensitivity to IR. Data of two independent experiments are shown for F1Pt, in each experiment an internal control (IC) was included. One experiment was performed for the family members (F1Si, F1Fa, F1Mo). (c and d) Fibroblasts from HC, F1Pt, and F02-98 were exposed to genotoxic inducers (0.2 µg/ml MMC, 20 J/m2 UV, or 10 Gy IR) in combination with a specific kinase inhibitor (2 µM DNA-PKi) (c) or only treated with 20 nM ATRi, 10 µM ATMi, or 2 µM DNA-PKi (d). Cell death was monitored by live imaging up to 120 h by quantifying the number of cells stained with SYTOX Green. Represented cell death kinetic plots are reflective of at least two independent experiments. (e and f) Corresponding proliferation curves of the data shown in Fig. 7, 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 (e) or with (f) specific kinase inhibitors (20 nM ATRi or 10 µM ATMi) as indicated. Percentage of confluency was monitored by live imaging up to 120 h. Data are representative of at least two independent experiments. (g) Schematics of genome editing of the ATR and ATRIP allele (top). Blue lines represent the location of crRNAs, orange arrows depict the joint primers annealing to sequence outside the crRNA cutting regions, and red arrows represent the primers annealing to depletion region, used for wild type (WT) allele detection. The western blot verification of mono-allelic cells is shown (bottom). Cells were seeded in 6 cm dish, and 1 μg/ml doxycycline was added in the culture medium 24 h before cell lysis to induce the expression of cas9 protein. (h) Protein expression of ATR, ATRIP, RPA1, RPA2, and TOPBP1 in the novel generated MCF10A mono-allelic cells. β-Actin was used as loading control. (i) Cell fitness of MCF10A mono-allelic cells with frameshift mutations serving as internal control. Each variant has three independent biological replicates. Mean and SEM are shown. *P < 0.05, **P < 0.01, ***<0.001 (two-tailed paired t tests). Source data are available for this figure: SourceDataFS5.

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