Fig. 5: PARP1 expression is repressed by a ROS/Rb pathway.

A MRC5 cells were infected with roGFP2-ORP1, a ratiometric ROS probe, encoding retroviral vector and a control or PLA2R1-encoding retroviral vectors and selected. Images were taken by Operetta system and quantified by Columbus software. The results are shown as representative from three experiments, mean ± SEM between three wells. P value was calculated using Student’s t-test ***p < 0.001. B MRC5 cells were infected with a control or a PLA2R1-encoding retroviral vector and selected. Cells were treated with NAC or Trolox daily during 3 days before analysis. Levels of PARP1 were quantified by RTqPCR and normalized against the level of GAPDH. The results are shown as mean ± SEM (n = 2). P value was calculated using Student’s t-test. *p < 0.05; **p < 0.01. C MRC5 cells where transfected with a control nontargeting pool (siCtrl) or siRNA pools targeting Rb. Two days after transfection cells were treated with H₂O₂; mRNA levels of Rb and PARP1 were quantified by RTqPCR 24 h later. Results were normalized against the level of GAPDH mRNA. The results shown are mean ± SEM (n = 4) and p value was calculated using Student’s t-test **p < 0.01; ***p < 0.001. D GSEA analysis was performed on PLA2R1 co-expressed genes. Graph displays negative enrichment of E2F target genes between PLA2R1 correlated genes (FDR q val < 0.001). E ChiP seq data³⁵ against Rb on senescent human fibroblasts were analyzed to reveal a binding of Rb to the PARP1 promoter. F MRC5 cells where transfected with a control nontargeting pool (siCtrl) or siRNA pools targeting Rb. The following day, cells were infected with a control or a PLA2R1-encoding retroviral vector and selected. mRNA levels of PLA2R1 (left), Rb (middle), and PARP1 (right) were quantified by RTqPCR. Results were normalized against the level of GAPDH mRNA. Mean ± SEM (n = 4) are shown and p value was calculated using Student’s t-test. *p < 0.05; ***p < 0.001.