Extended Data Fig. 6: Characterization of cytoplasmic DNA in senescent cells.

a, Left, quiescent and senescent cells were treated with BrdU as in Fig. 3c and the cellular localization of BrdU incorporation was visualized by immunofluorescence microscopy. Proliferating cells (EP (Prol)) are shown as a positive control for nuclear BrdU incorporation. Right, the signals were quantified using CellProfiler software (Methods). More than 200 cells were examined for each condition. b, Senescent (and early passage control) cells (neither labelled with BrdU) were fractionated into nuclear and cytoplasmic fractions, and the representation of L1 sequences in these compartments (as well as whole cells) was assessed with qPCR as in Fig. 3c (TaqMan multiplex qPCR assay¹⁶, amplicon F, Fig. 1b). Note that the y-axis units differ by tenfold between the left and right panels. c, Cells were examined by immunofluorescence microscopy for the presence of ORF1 protein, RNA–DNA hybrids, and ssDNA. See Methods and Supplementary Table 2 for antibodies. The RNA–DNA signal in senescent cells largely colocalized with the ORF1 signal and was lost after RNase A treatment. The ssDNA signal also colocalized with the ORF1 signal and was exposed by RNase treatment. The experiment was repeated three times with similar results. d, The pulled-down BrdU-containing DNA (a and Fig. 3c; Methods) was cloned and Sanger sequenced. Of the 96 total clones examined, 37 mapped to L1. Red boxes represent the relative positions of these clones on the L1 consensus sequence. e, Senescent cells labelled with BrdU (a and Fig. 3c) were immunoprecipitated with anti-BrdU antibodies, and the representation of L1 sequences in the pulled-down DNA was assessed using qPCR with primers spanning the entirety of L1 elements (Fig. 1b, c). f, Senescent cells were treated with L1 shRNA (using lentiviral vectors as described in Extended Data Fig. 5g) between 12 and 16 weeks of senescence, and expression of SASP genes was determined. g, Transcription throughout mouse L1 elements was assessed in a strand-specific manner using the same strategy as was applied to human L1 elements (Fig. 1b, c). The amplicons (designated W–Z to distinguish them from the human-specific primers) correspond to the 5′ UTR (W), ORF1 (X), ORF2 (Y) and 3′UTR (Z). See Methods and Supplementary Table 1 for primer sequences (primer sets 37 and 48–50). Poly(A) RNA was prepared from male white adipose tissue. A total of 12 mice were assessed (3 pools of 4 mice each) in 3 independent experiments. h, Expression of the three currently active families of mouse L1 elements (MdA, MdN and Tf). Primers were designed to distinguish 5′ UTR polymorphisms of the MdA, MdN and Tf families (Methods, Supplementary Table 1 primer sets 51–53). RT–qPCR was performed as in f (non-strand-specific). n = 3 independent biological samples, repeated in two independent experiments (a, b, e); n = 3 independent experiments (f). Data are mean ± s.d. *P ≤ 0.05, **P ≤ 0.01, one-way ANOVA with Tukey’s multiple comparisons test (a), unpaired two-sided t-test (b, e–h). Exact P values can be found in the accompanying Source Data.

Source Data