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. 2024 Jun 26;15:5423. doi: 10.1038/s41467-024-49740-w

Fig. 7. IFN-β induces replication stress and senescence in BJ fibroblasts.

Fig. 7

a BJ-RASV12 fibroblasts induced or not with 10 μg/ml doxycycline were treated or not with 300 U/ml of recombinant interferon-β (IFN-β) for a period of 8 days and the frequency of SA-β-gal positive cells was scored in two independent experiments. b BrdU incorporation in BJ-RASV12 cells treated or not with 50 or 300 U/ml IFN-β. Mean, SD and p values (two-sided unpaired t-test) are shown. At least 90 cells were scored per condition and in each experiment. Each point represent a biological replicate. c DNA fiber analysis of fork progression in BJ-RASV12 cells treated or not with 10 µg/ml Dox and 50 U/ml IFN-β, as described in Fig. 4a. Median values from two independent experiments are indicated. d Micronuclei frequency in BJ-RASV12 cells treated or not with 10 µg/ml Dox and 50 µ/ml IFN-β for 5 and 8 days. Mean values are shown for three independent experiments. e Number of 53BP1 foci in BJ-RASV12 cells treated or not with Dox and with 50 or 100 U/ml IFN-β for 5 or 8 days. Mean values are shown for three independent experiments. f IFN-β signaling contributes to RASV12-induced fork slowing. BJ-RASV12 fibroblasts were treated or not with 10 μg/ml doxycycline and 50 U/ml IFN-β for 6 days as indicated, in the presence of an anti-IFNα/β receptor antibody (α-IFNR) or a control IgG. Replication fork progression was measured by DNA fiber spreading. A minimum of 150 fibers were measured in each sample. Median track length is indicated in red. ****p < 0.0001, ns non-significant, two-tailed Mann–Whitney rank sum test. A representative experiment is shown (n = 2). g IFN-β signaling promotes RASV12-induced senescence. BJ-RASV12 fibroblasts treated as indicated above and β-galactosidase activity was analyzed on day 6. A minimum of 200 cells from 10 images were scored for SA-β-gal-positive cells in each sample. The percentage of SA-β-gal-positive cells (Mean ± SD) is shown for one representative experiment from two independent experiments (n = 2). ****p < 0.0001, two-tailed Mann–Whitney rank sum test. Source data are provided as a Source Data file.