Fig 1: Increased DSB and DDR are associated with cellular senescence in BRAP-deficient cells and cerebral cortices(A) Representative images of phospho-p53 immunofluorescence staining of WT and Brap-/- MEFs at P2.(B) Immunoblotting of total protein extracts from WT and Brap-/- MEFs at P1 and P3, respectively.(C) Immunoblotting of DNA damage response (DDR) proteins in WT and Brap-/- MEFs at P1.(D) Representative images of double immunofluorescence staining of WT and Brap-/- MEFs at P3 with antibodies against Ki67 (red) and 53BP1 (green).(E) Representative images of double immunofluorescence staining of WT and Brap-/- MEFs at P3 with antibodies against Ki67 (green) and ?H2A.X (red).(F) Immunoblotting analyses of histone extracts of MEFs at P1 or P3, showing increased ?H2A.X and ?H2A.X mono-ubiquitination in Brap-/- MEFs.(G) Kaplan–Meier curve shows significantly shortened lifespan (p < 0.0001 by log rank test) of BrapcKONPC mice (n = 46) relative to their littermate control mice (n = 29).(H) Representative images of senescence-associated ß-gal analysis of cerebral cortical sections from BrapcKONPC and control mice at three months of age. Note the pyramidal neuronal morphology of some SA-ß-gal + cells (red arrows) in high magnification views.(I) RT-qPCR shows an increased expression of p16Ink4a and Pai-1 (plasminogen activator inhibitor-1/Serpine1) in three-month-old BrapcKONPC cortices (Mean ± SD; n = 4 biological replicates). p-values calculated by Student’s t test are indicated.(J) Immunoblotting of cerebral cortical total protein extracts, demonstrating higher levels of senescence and SASP-like changes in the cortical tissue of BrapcKONPC relative to littermate control mice.(K and L) Immunoblotting of histone extracts from cortical tissue of three-month-old BrapcKONPC and control mice, showing elevated ?H2A.X as well as the presence of mono- and poly-?H2A.X ubiquitination in BrapcKONPC mice (boxed bands). K and L are the same immunoblot that was first probed by anti-?H2A.X, stripped, and re-probed by anti-ubiquitin.(M) Immunoblotting of total protein extracts from cortical tissue of BrapcKONPC and control mice, showing persistent elevation of 53BP1 in BrapcKONPC cortices from P8 (postnatal day 8) to 12M (12 months).(N) Representative images of ?H2A.X-NeuN double immunohistological staining of cerebral cortical sections from 4-month old WT or BrapcKONPC mice. The presence of ?H2A.X immunoreactivity in BrapcKONPC neurons is indicated by arrows.(O) Immunoblotting of total protein extracts from cortical tissue of BrapcKONPC or control mice at various ages, showing that Brap LOF does not increase cleaved caspase 3, an apoptosis marker. Embryonic Lis1+/- and Nde1-/- cortical tissues were used as positive controls for the presence of apoptosis. Nuclear DNA was stained with Hoechst 33,342. Bars: 50 um or as indicated.