Fig 2: The basal level of senescent cells is equal in the three groups of primary fibroblasts from adults. (A) Representative images of SA-ßgalactosidase assay for each group of age. 10 µM etoposide treatment was used as a positive control for senescence. (B) Quantification of positive cells in SA-ßgalactosidase assay with etoposide treatment or not. Five images were analyzed for each primary fibroblasts’ strains in the two conditions (mean ± SD; n = 5; * p < 0.5, ** p < 0.01). Exact p-values were determined using a paired Student’s t-test for comparison between cells treated with etoposide and cells without treatment. (C) Quantification of positive cells for 53BP1 staining. At least 200 cells from three images were analyzed for each primary fibroblast strain (mean ± SD; n = 5). (D) Representative images of 53BP1 immunostaining for each group of age. White arrows indicate positive cells. (E) Relative mRNA expression of P16 in primary fibroblasts. qPCR analysis was normalized to TBP and RPS17 housekeeping genes using the 2-??Ct quantification method (mean ± SD; n = 5). (F) P16 and P21 protein expressions were evaluated by immunoblotting in the three groups of primary fibroblasts (n = 5) and in a senescent positive control (cells treated with etoposide). One representative cell strains from each group of age is represented here. Actin protein expression is used as a loading control.

Fig 3: NFATC2 downregulation leads to cellular radiosensitivity. (A) NFATC2 mRNA levels were measured by RT-qPCR in three control fibroblast strains (HNF) infected with a lentiviral vector carrying either a shRNA scramble (sh-SCR) sequence or a shRNA targeting NFATC2 (sh-NFATC2). Results are mean +/- SD. The p-value was calculated using a Student’s t-test. Significant at *P < 0.05. (B) Representative image of immunoblotting analysis of NFATC2 protein expression in one control cell strain (HNF A) infected with lentiviral vectors sh-SCR or sh-NFATC2, and quantification (C). Results are mean +/- SD from immunoblotting analysis of three different cell strains infected with lentiviral vectors sh-SCR or sh-NFATC2. The p-value was calculated using a Student’s t-test. Significant at ***P < 0.001. (D) Lower SF2 was measured by colony survival assays in cells infected with lentiviral vector sh-NFATC2 compared to cells infected with lentiviral vector sh-SCR. Results are mean +/- SD from three cell strains. The p-value was calculated using a Student’s t-test. Significant at *P < 0.05. More numerous ?H2AX (E) and 53BP1 (F) residual foci, investigated by immunofluorescence, 24 h after 2 Gy irradiation in fibroblasts infected with lentiviral vector sh-NFATC2. Assessed in at least 100 cells in three cell strains infected with lentiviral vectors sh-SCR or sh-NFATC2. Results are mean +/- SD. The p-value was calculated using a two-way ANOVA. Significant at ****P < 0.0001.

Fig 4: Evaluation of radiation-induced DNA-double strand break foci using 53BP1. (A) Representative microscopy image showing (left to right) 53BP1, DAPI, merged image, and the resulting analyzed image after processing in ImageJ. (B–D) Number of 53BP1 foci at 2 h and 24 h after 3 Gy irradiation with FLASH (blue) or conventional dose rate (CONV, red) compared with controls (grey) for LU-HNSCC4 (B; 1,532 scored cells), MDA-MB-231 (C; 2,583 scored cells), and HeLasubclone (D; 2,973 scored cells). The box and whisker plots illustrate median (thick line), interquartile range (box) and the lowest/highest observation within ±1.5* interquartile range (IQR) from the box (whiskers). The individually scored cells are indicated with transparent circles. Data from two independent experiments.