Figure 5.
Absence of ATRIP does not abolish recruitment of ATR and its ability to phosphorylate substrates but reveals an insufficient ATR signaling response. (a) Fibroblasts from a HC, ATRIP patient (F1Pt), and ATR patient (F02-98) were left untreated or exposed to 0.02 µg/ml MMC, 200 J/m2 UV, or 2 Gy IR. ATR was stained by immunofluorescence after 24 h (MMC) or 3 h (UV and IR) after exposure and ATR nuclear foci were quantified. Dot plot represents pooled data from three independent experiments; at least 150 cells were analyzed for each condition. The median number of foci is depicted. ns: not significant, **P < 0.01, ***P < 0.001, ****P < 0.0001 (Kruskall–Wallis test and Dunn’s multiple comparisons test). (b) Protein expression of phosphorylation events (T1989-pATR, S317-pCHK1) and total protein (ATR, CHK1) 3 h after 200 J/m2 UV radiation. Immunoblotting was performed on fibroblasts from F1Pt, F02-98, and HCs (n = 3). Western blot is representative of three independent experiments. GAPDH serves as a loading control. (c) pATR and pCHK1 levels shown in Fig. 2 b were quantified. Bar graph depicts pATR and pCHK1 levels post UV treatment, expressed as a fold increase over the levels observed in the Mock condition. Mean and SD are shown. (d) Quantification of γH2AX nuclear fluorescence in HC and F1Pt fibroblasts 3 h after 200 J/m2 UV exposure and concomitant EdU pulse-labeling. The mean γH2AX intensity per nucleus is shown for EdU− and EdU+ fibroblasts. Dot plot represents pooled data from three independent experiments. At least 200 (EdU−) or 90 (EdU+) cells were analyzed per condition. The median value is depicted. ns: not significant, ****P < 0.0001 (multiple Mann–Whitney tests and Bonferroni–Dunn multiple comparisons test). Representative immunofluorescence images with DAPI, EdU, and γH2AX staining are shown (left). White arrows indicate EdU− cells. Scale bars are 20 µm. (e) γH2AX expression was determined by flow cytometric analysis 3 h following 200 J/m2 UV exposure in EdU− (G0/G1 and G2/M phase) fibroblasts of HC and F1Pt. Median fluorescence intensity (MFI) of γH2AX (AF488) is annotated on the histogram. Data are reflective of one experiment. Source data are available for this figure: SourceData F5. Refer to the image caption for details.

Absence of ATRIP does not abolish recruitment of ATR and its ability to phosphorylate substrates but reveals an insufficient ATR signaling response. (a) Fibroblasts from a HC, ATRIP patient (F1Pt), and ATR patient (F02-98) were left untreated or exposed to 0.02 µg/ml MMC, 200 J/m2 UV, or 2 Gy IR. ATR was stained by immunofluorescence after 24 h (MMC) or 3 h (UV and IR) after exposure and ATR nuclear foci were quantified. Dot plot represents pooled data from three independent experiments; at least 150 cells were analyzed for each condition. The median number of foci is depicted. ns: not significant, **P < 0.01, ***P < 0.001, ****P < 0.0001 (Kruskall–Wallis test and Dunn’s multiple comparisons test). (b) Protein expression of phosphorylation events (T1989-pATR, S317-pCHK1) and total protein (ATR, CHK1) 3 h after 200 J/m2 UV radiation. Immunoblotting was performed on fibroblasts from F1Pt, F02-98, and HCs (n = 3). Western blot is representative of three independent experiments. GAPDH serves as a loading control. (c) pATR and pCHK1 levels shown in Fig. 2 b were quantified. Bar graph depicts pATR and pCHK1 levels post UV treatment, expressed as a fold increase over the levels observed in the Mock condition. Mean and SD are shown. (d) Quantification of γH2AX nuclear fluorescence in HC and F1Pt fibroblasts 3 h after 200 J/m2 UV exposure and concomitant EdU pulse-labeling. The mean γH2AX intensity per nucleus is shown for EdU− and EdU+ fibroblasts. Dot plot represents pooled data from three independent experiments. At least 200 (EdU−) or 90 (EdU+) cells were analyzed per condition. The median value is depicted. ns: not significant, ****P < 0.0001 (multiple Mann–Whitney tests and Bonferroni–Dunn multiple comparisons test). Representative immunofluorescence images with DAPI, EdU, and γH2AX staining are shown (left). White arrows indicate EdU− cells. Scale bars are 20 µm. (e) γH2AX expression was determined by flow cytometric analysis 3 h following 200 J/m2 UV exposure in EdU− (G0/G1 and G2/M phase) fibroblasts of HC and F1Pt. Median fluorescence intensity (MFI) of γH2AX (AF488) is annotated on the histogram. Data are reflective of one experiment. Source data are available for this figure: SourceData F5.

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