Prideapplication/xmlftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/08/PXD063287/HUMAN-cannonical-240119.fastaftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/08/PXD063287/250218_Maricarmen_PHS_vesicles_RAW.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/08/PXD063287/20250223_131005_250223_Maricarmen_PHS_Vesiculas.sneprimary200OKroman.gonzalez@cabimer.esRomán González-PrietoMass SpectrometryData-independent acquisitionRadioadaptative response/bystander effect/dna end resection/dna repairhttps://www.ebi.ac.uk/pride/archive/projects/PXD063287EVs Mass Spectrometry sample preparation. Purified EVs were resuspended in RIPA lysis buffer (Thermo Scientific) by gently pipetting and vortexing. Then, lysates were incubated with 10 mM TCEP (Merck) for 45 min at 37ºC with shaking to promote disulfide reduction, followed by the cysteine alkylation with 40 nM cloracetamide (Merck) for 30 min at room temperature in the dark. Meanwhile, beads were prepared using paramagnetic bead technology based on the Single-Pot Solid-Phase-Enhanced Sample Preparation (SP3), in which two types of SpeedBeads, mixed at a 1:1 ratio, were used: 45152105050250 and 65152105050250 (Cytiva). Samples were incubated with the beads mixture and 100% acetonitrile (Merck) with shaking for 35 min at room temperature. Proteins were then washed twice with 70% ethanol (Merk), followed by two additional washes with 100% acetonitrile. Finally, the beads were incubated with 250 ng trypsin in 50 mM ammonium bicarbonate at 37ºC with shaking overnight. Digested peptides were separated from the beads using a 0.45µm centrifugal filter unit (Millipore) and acidified by adding 2% trifluoroacetic (TFA) acid. Subsequently, peptides were desalted and concentrated on triple-disc C18 Stage-tips as previously described (Rappsilber). Stage-tips were in-house assembled using 200 µL micro pipet tips and C18 matrix (Sigma-Aldrich). Stage-tips were first activated by passing through 100 µl of methanol. Next, 100 µl of Buffer B (80% acetonitrile, 0.1% formic acid), 100 µl of Buffer A (0.1% formic acid), the acidified peptide sample, and two times 100 µl Buffer A were passed through the Stage-tip. Elution was performed twice with 30 µl of Elution buffer (32,5% acetonitrile, 0.1% formic acid solution). Samples were vacuum dried using a Universal Vacuum System UVS400S coupled to a SpeedVac SPD121P (Thermo) and stored at −20 °C. Prior to mass spectrometry analysis, samples were reconstituted in 20 µl 0.1% formic acid and transferred to autoload vials. Mass Spectrometry Data acquisition Mass spectrometry-based proteomics data was acquired at the Proteomics facility in Institute of Biomedicine of Seville (IBiS). As previously done (Salas-Lloret et al.), samples were measured using a nanoElute II LC system coupled to a timsTOF SCP mass spectrometer with an electrospray source (Bruker Daltonics). LC separations were performed on C18 HPLC column (Aurora 25 cm and 75 µm ID, IonOpticks) kept at 50°C. Gradient elution was performed with a binary system consisting of (A) 0.1% aqueous formic acid and (B) 0.1% formic acid in Acetonitrile. An increasing linear gradient (v/v) was used (t (min), %B): (0, 2); (40, 17); (60, 25); (66, 37); (67, 95), followed by an equilibration step. Mass spectrometric analysis was performed in a data independent acquisition parallel accumulation serial fragmentation (dia-PASEF) mode, with 100–1700 m/z mass range, an ion mobility range from 0.64 to 1.45 V s cm−2, capillary voltage set to 1500 V, an accumulation and ramp time at 100 ms and the collision energy as a linear ramp from 20 eV at 1/K0 = 0.6 V s cm−2 to 59 eV at 1/K0 = 1.6 V s cm−2.PrideMass Spectrometry Data analysis Mass spectrometry raw dia-PASEF data was analyzed using Biognosys Spectronaut (v19.5.241126) using a directDIA+ search with modified BGS Factory settings. Predicted spectral library was built using a FASTA file corresponding to the reference human proteome (Uniprot 19th January 2024) only including canonical proteins (20,413 entries). Maximum number of variable modification were set to 2 and Carbamidomethyl (C) was disabled as a fixed modification. A pivot report was generated and further processed in the Perseus Computational Platform (Tyanova et al 2016). Proteins not identified in every replicate for at least one condition were removed and missing values were randomly imputed using normally distributed values with 0.3 width and 1.8 down shift considering the total matrix. Statistical analysis was performed using two-sided Student’s t tests and exported to MS Excel for comprehensive browsing of the data.ProteomicsRomán González-PrietoCABIMER, ESHomo Sapiens (human)PARTIAL10.1093/NAR/GKAG764roman.gonzalez@cabimer.esAndalusian Center for Regenerative Medicine and Cell Biology, CABIMER, University of SevilleSpainfalsemiRNA-mediated cell-to-cell communications boost DNA repair during the Radioadaptative Response in human fibroblastsThe radioadaptive response is a phenomenon in which cells exposed to a low priming dose of ionizing radiation develop increased resistance for a period of time to a subsequent higher challenging dose. Little is known of the molecular components of this response. Here, we focus on determining if during the RAR there are changes in the Homologous Recombination, the more faithful DNA Double Strand break repair pathway. Using immortalized fibroblasts as a model system, we demonstrate that primed cells exhibit faster DNA resection, the initial step of homologous recombination, following a challenging dose, as indicated by RPA foci kinetics. We further establish that the stimulation of resection during the RAR is mostly established and maintained through a bystander effect mediated by cell-to-cell communication via extracellular vesicles. Indeed, media exchange experiments demonstrated that soluble factors in the conditioned media were sufficient to fully recapitulate the RAR effect. RNA analyses of extracellular vesicles cargo revealed differential expression of miRNAs, particularly miR-126-3p and miR-451a, which we show play crucial roles in modulating the RAR. Notably, inhibition of miR-451a was sufficient to induce the RAR in cells that typically do not exhibit this response. Finally, we identified the p38-CCAR2 axis as a critical regulator of the RAR-mediated DNA end resection boost. p38 activation following priming irradiation block CCAR2 activity, facilitating a more efficient repair process. Collectively, our findings elucidate a novel mechanism by which EV-mediated communication and specific miRNA signaling enhance HR efficiency in response to radiation, offering insights into potential therapeutic applications in radiotherapy.2026-08-032025-04-24PXD063287NEWT:3555NEWT:71647NEWT:330879NEWT:35554NCBITaxon:12939NEWT:32046NEWT:544496NEWT:59529NEWT:2042546NEWT:32049NEWT:1392696NEWT:259447NEWT:295546NCBITaxon:2719036NEWT:45351NEWT:445974NEWT:43179NEWT:180454NEWT:5722NCBITaxon:1280NEWT:112503NEWT:1129NEWT:309807NEWT:10245NEWT:55153NEWT:309800NEWT:281395NEWT:1211601NEWT:876138NEWT:44271NEWT:193516NEWT:351581NEWT:43186NEWT:428146NEWT:498257NEWT:10036NEWT:1590NEWT:498019NEWT:1351NEWT:1438992NEWT:1352NEWT:2649997NEWT:1147161NEWT:661410NEWT:638632NEWT:263737NEWT:224326NEWT:1333499NEWT:376619NCBITaxon:79857NEWT:1096976NEWT:5702NEWT:95648NEWT:1589NEWT:135622NEWT:1349NEWT:35786NEWT:1580NEWT:399784NEWT:96731NEWT:383379NEWT:1401257NEWT:418106NEWT:10029NEWT:913645NEWT:44491NEWT:641809NEWT:27933NEWT:556484NEWT:317447NEWT:4688NEWT:7955NEWT:7719NEWT:7959NEWT:2261NEWT:868565NEWT:31156NEWT:3112NEWT:79329NEWT:4442NEWT:192875NEWT:1214915NEWT:12637NEWT:59729NEWT:2164133NEWT:284218NEWT:295105NEWT:108061NEWT:60711NEWT:1316931NEWT:224308NEWT:3347NEWT:511145NEWT:160621NEWT:212790NEWT:931281NEWT:4432NEWT:5762NEWT:5763NEWT:658457NEWT:1310161NEWT:77133NEWT:295358NEWT:145481NCBITaxon:79824NEWT:2246NEWT:2652724NEWT:1912919NEWT:5757NEWT:68415NCBITaxon:4563NEWT:5755NEWT:44689NEWT:44447NEWT:752555NEWT:498211NEWT:5759NEWT:347256NEWT:1736231NEWT:5518NEWT:398007NEWT:1527468NEWT:1392NEWT:498217NEWT:498216NEWT:2242NEWT:11320NEWT:286NEWT:391619NEWT:246196NEWT:287NEWT:246197NEWT:1367269NEWT:145458NEWT:633149NEWT:1149786NEWT:10239NEWT:68895NEWT:44685NEWT:161934NEWT:4897NEWT:1148NEWT:5744NEWT:213863NEWT:5508NEWT:3329NEWT:5507NEWT:410661NEWT:55571NEWT:35500NEWT:1235816NEWT:1140NCBITaxon:2157NEWT:1143NEWT:4896NEWT:1287689NEWT:1390NEWT:11557NEWT:1094343NEWT:1462472NEWT:1336795NEWT:644042NEWT:294NEWT:1773NEWT:1182590NEWT:3712NEWT:82380NEWT:3711NEWT:935293NEWT:375146NEWT:270643NEWT:2065263NEWT:177437NEWT:1772NEWT:10418NEWT:118698NEWT:1616117NEWT:263NEWT:118696NEWT:34865NEWT:52283NEWT:11988NEWT:284812NEWT:8175NEWT:400667NEWT:43330NEWT:1603293NEWT:408169NEWT:44664NEWT:47946NEWT:459758NEWT:3702NEWT:1245466NEWT:243277NEWT:1246791NEWT:7067NEWT:118694NEWT:586722NEWT:2850NEWT:118691NEWT:34871NEWT:33548NEWT:274NEWT:7070NCBITaxon:287NEWT:408172NEWT:408170NEWT:96794NEWT:3708NEWT:332648NEWT:44670NEWT:536231NEWT:376219NEWT:219813NEWT:1510NEWT:1436733NEWT:460519NEWT:1247411NEWT:1515NEWT:572307NEWT:1432138NEWT:1424507NEWT:1194599NEWT:272844NEWT:9483NEWT:333760NEWT:56636NEWT:1513458NEWT:1233681NCBITaxon:40559NEWT:506599NEWT:2853422NEWT:81077NEWT:84588NEWT:1679718NEWT:480NEWT:65349NEWT:67767NEWT:46835NEWT:109757NEWT:582580NEWT:300852NEWT:1502NEWT:128017NEWT:376686NEWT:95486NEWT:508771NEWT:1883446NEWT:253NCBITaxon:2759NEWT:1233435NEWT:93061NEWT:93062NEWT:34613NCBITaxon:4932NEWT:109760NEWT:943274NEWT:644223NEWT:235443NEWT:108458NEWT:5936NEWT:272623NEWT:272624NEWT:320637NEWT:886559NEWT:3750NEWT:55521NEWT:983964NEWT:118499NEWT:11706NEWT:32644NEWT:527796NEWT:1130798NEWT:55529NEWT:499175NEWT:109779NEWT:3745NEWT:1715989NCBITaxon:4751NEWT:476272NEWT:3747NEWT:195051NEWT:3988NEWT:1116234NEWT:561007NEWT:1255228NEWT:649908NEWT:410289NEWT:373153NEWT:3983NEWT:203696NEWT:352472NEWT:1071661NEWT:1202532NEWT:360094NEWT:470NEWT:41364NEWT:5911NEWT:1313NEWT:1218097NEWT:1080348NCBITaxon:50557NEWT:39251NEWT:39491NEWT:212142NCBITaxon:5811NEWT:29491NEWT:101841NEWT:153481NEWT:2014887NEWT:33952NEWT:153009NEWT:261756NEWT:571256NEWT:40483NEWT:63366NEWT:63367NEWT:688333NEWT:215402NCBITaxon:548681NEWT:318586NCBITaxon:5820NEWT:1547NEWT:272634NEWT:198107NEWT:9031NEWT:27292NEWT:1872122NEWT:1308NEWT:7091NEWT:1034015NEWT:760568NEWT:141262NEWT:108931NEWT:1293497NEWT:1055524NEWT:4087NEWT:9778NEWT:306NEWT:150475NEWT:303NEWT:267872NEWT:97477NEWT:7111NEWT:347515NEWT:172269NEWT:9534NEWT:6269NEWT:5180NEWT:256737NEWT:4090NEWT:4092NEWT:9541NEWT:8694NEWT:4093NEWT:33936NEWT:4096NEWT:8692NEWT:2903NEWT:185579NEWT:941442NEWT:536088NEWT:13076NEWT:1226408NEWT:7108NEWT:40479NEWT:43989NEWT:4076NEWT:142615NEWT:317NEWT:1006581NEWT:33940NEWT:885318NEWT:39488NEWT:550NEWT:4081NEWT:273068NEWT:554NEWT:98334NEWT:451516NEWT:552NEWT:4084NEWT:1325291NEWT:63577NEWT:36185NEWT:1225786NEWT:7574NEWT:1715256NEWT:30640NEWT:575412NEWT:929793NEWT:1507179NEWT:29204NEWT:28112NEWT:114155NEWT:6494NEWT:6491NEWT:507601NEWT:520NEWT:186441NEWT:634955NEWT:643680NEWT:214092NCBITaxon:6157NEWT:6239NEWT:13095NEWT:162425NEWT:216257NEWT:102169NEWT:208893NEWT:9986NEWT:4054NEWT:8654NEWT:8658NEWT:1268063NEWT:8657NEWT:8655NEWT:5147NEWT:98351NEWT:1263854NEWT:118503NEWT:2059687NEWT:160488NEWT:28104NEWT:207559NEWT:407821NCBITaxon:2NEWT:85057NEWT:985076NEWT:568708NEWT:1215323NEWT:986NEWT:52641NEWT:7159NEWT:267671NEWT:28532NEWT:353152NEWT:8496NEWT:7165NEWT:1257118NEWT:27448NEWT:40674NEWT:1194669NEWT:51329NEWT:243230NEWT:443906NEWT:1080772NEWT:519NEWT:8479NEWT:8485NEWT:6063NEWT:49240NEWT:1328388NEWT:104395NEWT:667127NEWT:9557NEWT:9315NEWT:6289NEWT:377586NEWT:6287NEWT:38569NEWT:300641NEWT:727NEWT:9796NEWT:9554NEWT:118562NEWT:725NEWT:7137NEWT:469008NEWT:256318NEWT:9321NEWT:206411NC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