ATRIP deficiency disrupts ATR signaling, DNA replication, cell cycle progression, and T cell proliferation . (a) Representative immunofluorescence images of cells shown in Fig. 5 a with DAPI and ATR staining. 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 following 24 h (MMC) or 3 h (UV and IR) of treatment. Images are representative of three independent experiments. Scale bars are 20 µm. (b) pATR and pCHK1 levels shown in Fig. 5 b were quantified and represent three independent experiments. Bar graph depicts pATR and pCHK1 levels post 200 J/m² UV treatment, expressed as a fold increase over the mean levels observed in three HCs. Mean and SD are depicted. (c) Immunoblotting of T1989-pATR and total ATR with and without lambda phosphatase (λPPase) treatment on HC fibroblasts, untreated or 3 h after 200 J/m² UV exposure. GAPDH serves as a loading control. (d) Cell cycle distributions of PHA blasts from HCs and ATRIP patient (F1Pt). Cells were either untreated or treated with 0.02 µg/ml MMC and subsequently harvested at the timepoints indicated on the schematics. Scatter dot plot depicts data from at least five independent experiments. Mean and SD are shown. ns: not significant, ***P < 0.001 (multiple paired t tests). (e) Representative immunofluorescence images with DAPI, EdU, and RPA staining of data shown in Fig. 6 d. HC and F1Pt fibroblasts were untreated or exposed to 1 mM HU for 3 h. Images are representative of three independent experiments. Scale bars are 20 µm. (f) Flow cytometric (FCM) EdU pulse-labeling profiles of HC and F1Pt fibroblasts demonstrating the inhibiting effect of HU treatment on replication progression. Cells were exposed to 1 mM HU for 3 h and subsequently harvested. Data are representative of two experiments. (g) EdU pulse-chase analysis after exposure to 4 Gy IR in the absence or presence of 20 nM ATRi. HC and F1Pt PHA blasts were harvested after 9 h. Bar plot (left) shows EdU⁺ cells present in G2/M phase after IR exposure, depicted as a fold change over the percentage observed in the mock condition. Data represent five independent experiments. Mean and SD are shown. **P < 0.01 (two-tailed paired t test). Bar plot (right) shows percentages of EdU⁺ cells present in G0/G1, S, and G2/M phase. Data from one experiment are shown. (h) FCM gating strategy of EdU pulse-chase kinetics presented in Fig. 6 g and Fig. S4 g. (i) PF and PI of CD8⁺ and CD4⁺ PHA blasts from HC and F1Pt. Cells were labeled with CTV and subsequently cultured for 96 h in the presence or absence of 0.02 µg/ml MMC. Data of at least two independent experiments are shown, with each data point representing one biological replicate. Mean and SD are shown. ns: not significant, *P < 0.05, **P < 0.01, ****P < 0.0001 (multiple paired t tests). Source data are available for this figure: SourceDataFS4.