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. 2017 May 16;8:15287. doi: 10.1038/ncomms15287

Figure 5. Overexpression of Dnase2a attenuated the effects of Alix or Rab27a knockdown in HDFs.

Figure 5

Pre-senescent TIG-3 cells were infected with retrovirus encoding flag-tagged Dnase2a (lanes 4–6) or empty vector (lanes 1–3). After selection with puromycin, cells were transfected with indicated siRNA oligos and then subjected to western blotting using antibodies shown right (a), NanoSight analysis (NTA) for quantitative measurement of isolated exosome particles and western blotting using antibodies against canonical exosome markers shown right (exosome) (b), isolation of cytoplasmic fraction followed by quantitative PCR (qPCR) analysis of chromosomal DNA (c), immunofluorescence staining for markers of DNA damage (γ-H2AX [red], pST/Q (green) and 4′,6-diamidino-2-phenylindole (blue)) (d), qPCR analysis of IFNβ gene expression (e), analysis of intracellular ROS levels (e) or to apoptosis analysis at day 4 (e). Tubulin was used as a loading control (a). The histograms indicate the percentage of nuclei that contain more than 3 foci positive for both γ-H2AX and pST/Q staining (d). At least 100 cells were scored per group (d). The representative data from three independent experiments are shown. For all graphs, error bars indicate mean±s.d. of triplicate measurements. (**P<0.01. ***P<0.001; one-way analysis of variance).