Fig 2: Cytoplasmic DNases prevents cytoplasmic accumulation of nuclear DNA and SASP. a–f Pre-senescent TIG-3 cells were infected with retrovirus encoding flag-tagged DNase2 (lanes 3 and 4), TREX1 (lane 7 and 8) or empty vector (lanes 1, 2, 5, and 6). After selection with puromycin, cells were super-infected with retrovirus encoding HRasV12 and subjected to western blotting using antibodies shown right (a), qPCR analysis for cytoplasmic accumulation of nuclear DNA (b), RT-qPCR analysis of SASP factor gene expression (c), analysis of intracellular ROS levels (d), immunofluorescence staining for markers of DNA damage (?-H2AX and pST/Q substrate) (e) or BrdU incorporation analysis (f). Tubulin was used as a loading control (a). The histograms indicate the percentage of nuclei that contain more than 3 foci positive for both ?-H2AX and pST/Q staining (e). The representative data from three independent experiments are shown. For all graphs, error bars indicate mean ± standard deviation (s.d.) of triplicate measurements. (**P < 0.01. ***P < 0.001; one-way ANOVA)

Fig 3: E2F-dependent downregulation of DNase2 and TREX1 in senescent cells. a, b Pre-senescent TIG-3 cells were rendered senescent by either serial passage (late passage) or ectopic expression of oncogenic ras (+HRasV12). These cells were then subjected to RT-qPCR analysis of DNase2 and TREX1 gene expressions (a), or incubated with 10 µM MG132 for 12 h, then subjected to western blotting using antibodies shown right (b). c, d Pre-senescent TIG-3 cells were infected with retrovirus encoding shRNA against DP1 or control three times. After puromycin selection, these cells were then subjected to RT-qPCR analysis of indicated genes shown right (c) or western blotting using antibodies shown right (d). Pre-senescent TIG-3 cells were subjected to ChIP analysis using antibodies shown at top and PCR primers of DNase2 promoter or TREX1 promoter shown as red arrows in the scheme (e, f). Tubulin was used as a loading control (b, d). The representative data from three independent experiments are shown. Error bars indicate mean ± standard deviation (s.d.) of triplicate measurements. (***P < 0.001; one-way ANOVA)

Fig 4: Overexpression of Dnase2a attenuated the effects of Alix or Rab27a knockdown in HDFs.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).