Fig. 4: Induction of a pro-inflammatory senescence phenotype in RPTECs and HFFs upon HCMV infection.

a GSEA of the comparison between HCMV-infected and mock-infected cells, showing the enrichment of senescence genes associated with the SenMayo signature⁴³ in RPTECs and HFFs. The enrichment score curve is depicted in green, with genes on the far left (red) correlating with HCMV-infected cells, and genes on the far right (blue) correlating with mock-infected cells. The vertical black lines indicate the position of each gene in the gene set analyzed. NES and FDR are shown. b Representative images of senescence-associated β-gal (SA-β-gal) staining in HCMV- or mock-infected RPTECs, ARPE-19 and HFFs at 3dpi (MOI 1, 3 and 0.5, respectively). The blue areas represent positive SA-β-gal staining. Scale bars, 250 μm. c Histograms representing the percentage of SA-β-gal⁺ RPTECs, ARPE-19 and HFFs. Data are expressed as mean values ± SD from three independent experiments (**P < 0.01, ***P < 0.001; multiple unpaired t-test). d RPTECs, ARPE-19 and HFFs were infected as described in (b). Subsequently, cells were incubated with EdU for 20 h and stained as described in the Material and Methods section. DAPI was used to counterstain the nuclei. Scale bars, 200 μm. e Histograms representing the percentage of EdU⁺ cells. Data expressed as mean values ± SD from three independent experiments (*P < 0.05; multiple unpaired t-test). f The same images used in (d) were also used to calculate the total number of cells per field (*P < 0.05, **P < 0.01; multiple unpaired t-test).