Fig 2: LINC01468 impedes PAI1 protein degradation via interaction with USP5.A Protein levels of PAI1 in response to the knockdown or overexpression of LINC01468 or SERBP1 were measured via western blot. B After CHX treatment, western blot analysis was done to measure protein levels of PAI1 in cells with or without LINC01468 knockdown. C IP-WB was done for assessment of PAI1 ubiquitination under LINC01468 silence. D After RNA pull-down assay, western blot was done to detect USP5 level pulled down by indicated probes. E The binding correlation between LINC01468 and USP5 was verified via RIP assay. F Co-IP assay was applied for evaluation of the interaction between USP5 and PAI1. G The changes in USP5 mRNA and protein levels in cells transfected with si-USP5 were assessed via RT-qPCR and western blot. H The influence of USP5 silence on PAI1 level was evaluated via western blot. I IP-WB was applied for assessment of PAI1 ubiquitination under the influence of USP5 depletion. Results were exhibited as the mean ± SD on the basis of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 indicated the statistical significance of experiments data.

Fig 3: a Confocal imaging shows the alignment of gCapA and gCapD in the lungs.b Dot plot showing the top 60 genes that differentiate each of the general capillary phenotypes (adjusted p-value < 0.05, Two-sided Wilcoxon rank sum test with Bonferroni correction).c Western Blots of gCaps A-E and Aerocytes showing ZO-1(Zonula occludens-1), FosB (FosB Proto-Oncogene, AP-1 Transcription Factor Subunit), eNOS(endothelial Nitric oxide synthase), Occludin, PAR3 (Proteinase-activated receptor 3), E-Selectin, P-Selectin, TGFBR1 (transforming growth factor-beta (TGF-ß) receptor type 1), TGFBR3 (transforming growth factor-beta (TGF-ß) receptor type 3), Afadin, Gja10 (Gap Junction Protein Alpha 10), iNOS (inducible nitric oxide synthase), PAI-1(plasminogen activator inhibitor 1), ICAM1 (Intercellular Adhesion Molecule 1), L-Selectin, TPA (Tissue plasminogen activator), VWF (von Willebrand factor), PAR1 (proteinase-activated receptor 1)

Fig 4: 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.