Extended Data Fig. 1: Additional biological properties of retroviral VIS.

a, SA-β-gal staining, SAHF formation by DAPI, and p16^INK4A staining of HDFs (Tig3 and WI38) five days after retroviral infection or mock control. Representative photomicrographs of n = 3 independent experiments. b, Gene set enrichment analysis (GSEA) of virus infection-relevant GO terms probing RNA-seq datasets of WI38 HDF in VIS and OIS. Positive normalized enrichment scores (NES) indicate enrichment in VIS (dark grey bars) or OIS (light grey bars) compared to proliferating counterparts (mock infection for VIS, or empty vector control for OIS). NES with FDR q ≤ 0.1 are considered statistically significant and presented (for individual q-values, see Supplementary Information); n = 3 biological replicates each. c, Gene expression analysis for core senescence and SASP genes by RT-qPCR in WI38 and Tig3 as in a. Mean relative transcript level compared to mock control + s.d. of n = 3 independent experiments are shown. d, Growth curve analyses of HDF infected with retrovirus at different MOI as indicated, showing that high-titer virus induced VIS, reflected by stable cell numbers over time, while lower virus titer remained compatible with exponential cell growth. n = 3 independent experiments are presented as mean cell numbers ± s.d. e, SA-β-gal staining (left) and gene expression analysis of the indicated transcripts by RT-qPCR (right) in wild-type (WT) or senescence-defective p53^-/- MEF, five days after infection with high-titer retrovirus or mock as a control. Representative photomicrographs with fractions of SA-β-gal-positive cells, and mean relative transcript levels normalized to mock control ± s.d. of n = 4 independent experiments are shown. f, Gene expression analysis of the indicated transcripts by RT-qPCR in IMR90 cells expressing JMJD2C, p53 shRNA (shp53), or control vector as in Fig. 1e. g, Multiplex bead-based protein analysis of SN of senescence-incapable IMR90 as in Fig. 1e. SN_mock = SN of mock-infected cells; SN_virus = SN of retrovirus-infected cells. Mean expression levels of n = 3 biological replicates are shown. h, cGAS/STING activation upon viral infection, as evidenced by a higher induction of cyclic guanosine monophosphate-adenosine monophosphate (2'3'-cGAMP) by ELISA analysis in matched pairs of HDF after either mock or retrovirus infection (mean of n = 4 independent experiments for each cell line, upper panel). Mean cGAMP levels + s.d. for senescence-incapable IMR90 as in Fig. 1e (n = 4 independent experiments, lower panel). i, SA-β-gal staining of IMR90 cells, treated with reverse transcriptase inhibitor azidothymidine Zidovudine (50 µM), cGAS inhibitor G150 (5 µM), STING inhibitor H-151 (1 µM), or DMSO. Mock infection and DMSO solvent treatment as negative controls. Representative photomicrographs and quantification of positively stained cells as mean ± s.d. of n = 3 independent experiments are shown. j, 2'3'-cGAMP ELISA analysis of VIS IMR90 cells as in i .