Day 4 (e,f). The representative data from three independent experiments are shown. For all graphs, error bars indicate mean .d. of triplicate measurements. (Po0.01, Po0.001; one-way analysis of variance).these information strongly suggested that exosome secretion plays a key function in the maintenance of cellular homeostasis by preventing the aberrant activation of DDR pathways, at least in certain types of typical human cells. Exosomes excrete harmful cytoplasmic DNA from cells. To additional discover this notion, we analysed how exosome secretion prevents the aberrant activation of DDR pathways. In looking for an explanation, we noted that exosomes released from HDFshave the potential to activate the DDR pathway in recipient pre-senescent HDFs, based on the quantity of added exosome (Supplementary Fig. 5). This result led us to propose that exosome secretion may possibly stop the aberrant activation from the DDR pathway, by excreting harmful cellular constituents from cells. Exosomes are known to include several cellular elements, including proteins, lipids, RNA and DNA214,43. Amongst them, DNA is especially exciting, simply because fragmented DNA is recognized to activate the DDR in normalNATURE COMMUNICATIONS | 8:15287 | DOI: 10.1038/ncomms15287 | nature.com/naturecommunicationsARTICLEasiRNA:(kDa) 16 46 78 33 78 33 33NATURE COMMUNICATIONS | DOI: 10.1038/ncommsEarly passageaRelative quantity of cellsbsiRNA: 1. Manage two. Alix three. Isoproturon Cancer Rab27atro l Al ixRelative amounts of apoptotic cellsP-p53 Ser15 Alix Rab27a Tubulin CD63 CD81 Tsg1.five 1 0.five 0 1 two three 4 5 six Days15 ten 5CWCLRelative volume of Exosome exosomes/celldDNA damage foci positive cells ( ) siRNA: Manage Alix Rab27a 80 60 40 20 0 1 21 NTA 0.ten m10 m10 m2 + + three + + + + + + Alix TubulinH2AX pST/Q DAPIesiRNA:Manage Alix Empty vector si-res.Alix cDNA WCL(kDa) 78 78 2 1.5 1 0.five 0 40 30 20 ten 0 80 60 40 20Control Rab27a Empty vector si-res.Rab27a cDNAfsiRNA:+ + + + + ++ + Rab27a Tubulin(kDa)WCL33Relative amounts Relative level of of apoptotic cells exosomes/Lactacystin Description cellRelative amounts Relative level of of apoptotic cells exosomes/cellNTA1.5 1 0.five 0 30 20 10 0 60 40 20 0 1 2 3NTADNA damage foci constructive cells ( )DNA damage foci positive cells ( )Figure 2 | Inhibition of exosome secretion in pre-senescent HDFs. (a) Pre-senescent TIG-3 cells had been subjected to transfection with indicated siRNA oligos twice (at two day intervals). These cells had been then subjected to western blotting applying antibodies shown proper (WCL) or to exosome isolation followed by western blotting using antibodies against canonical exosome markers shown suitable (exosome) and NanoSight evaluation (NTA) for quantitative measurement of isolated exosome particles. The representative data from 3 independent experiments are shown. Tubulin was utilised as a loading manage. (b ) Pre-senescent TIG-3 cells cultured beneath the situations described inside a had been subjected to cell proliferation analysis (b), apoptosis analysis at day four (c) or to immunofluorescence staining for markers of DNA damage (g-H2AX [red], phosphor-Ser/Thr ATM/ATR (pST/Q) substrate [green] and 40 ,6-diamidino-2-phenylindole [blue]) (d). The representative data from 3 independent experiments are shown. The histograms indicate the percentage of nuclei that contain far more than 3 foci good for each g-H2AX and pST/Q staining (d). At the very least one hundred cells had been scored per group (d). (e,f) Pre-senescent TIG-3 cells have been infected with retrovirus encoding flag-tagged wild-type Alix or Rab27a protein containing a mutated siRN.