Fig. 10: HCMV infection drives an IL-6-mediated senescence/inflammatory loop in RPTECs that is extended to bystander non-infected cells.

a Schematic representation of the workflow followed to assess the occurrence of paracrine senescence in RPTECs, ARPE-19, and HFFs, as described in the Materials and Methods section. Created with BioRender.com. b Representative images of EdU incorporation staining in RPTECs, ARPE-19, and HFFs. Cells were treated for 2 days with UVB-irradiated conditioned media harvested from to the corresponding mock- or HCMV-infected cells at 3 dpi. After treatment, cells were incubated with EdU for an additional 24 h (3 days of total treatment) and stained as described in the Material and Methods section. DAPI was used to counterstain the nuclei. Scale bars, 200 μm. c, d Histograms representing the percentage of EdU-positive cells (c) or the total number of cells per field (d) upon treatment with UVB-exposed conditioned media from mock- or HCMV-infected cells. Three random fields of view per cell line from 3 different experimental replicates (at ×63 magnification) were captured. Data are expressed as mean values ± SD of three independent experiments (*P < 0.05, **P < 0.01; multiple unpaired t-test). e The conditioned medium obtained from infected RPTECs as described in a and b was used to treat fresh RPTECs in the presence of TCZ (25 μg/mL) for 48 h and then incubated with EdU for an additional 24 h, resulting in a total treatment period of 3 days. Vehicle (DMSO)-treated cells were used as control. Scale bars, 200 μm. f, g Histograms representing the percentage of EdU-positive cells (f) or the total number of cells per field (g) following exposure to vehicle- or TCZ-treated UVB-exposed conditioned media from mock- or HCMV-infected RPTECs. Three random fields of view per cell line from 3 different experimental replicates (at ×63 magnification) were captured and analyzed. Data are expressed as mean values ± SD of three independent experiments (*P < 0.05; multiple unpaired t-test). h RPTECs were infected with HCMV (MOI 1) and cultured for 3 days with the conditioned media generated as described in a (left panel) or adding fresh medium after the first 24 hpi (right panel). The results representing the percentage of IEA-positive infected RPTECs, normalized to the total number of DAPI-positive cells, were plotted. Image analysis was conducted using LAS X software (Leica Microsystems) and values are expressed as mean ± SD (error bars) relative to six different experiments (**P < 0.01; multiple unpaired t-test).