Research Paper Volume 16, Issue 20 pp 12977—13011

Figure 7. Catalytically inactive WRN mutants restore nuclear LBR levels and SATII silencing in wrn null cells. (A) Stable expression of the wild type, exonuclease-dead (E84A), or helicase-dead (K577M) WRN in Δwrn-3 cells was measured by IF in situ with an antibody against WRN. A dotted line marks the cutoff MFI below which cells were considered negative for WRN expression. (B) Nuclear levels of LBR were measured by IF in situ in the same cells as in (A). For WRN-complemented cells, only WRN-positive cells measuring above the cutoff line in (A) for WRN expression were plotted. The graph represents three independent experiments for the wild type and E84A WRN and two independent experiments for the K577M WRN. (C) Quantitations of the indicated PLA analyses performed in the Δwrn-3 GM639 with or without expression of the wild type WRN transgene. Crossbars in graphs are distribution means. (D) RT-qPCR analyses of SATII satellite repeat transcription performed on Δwrn-3 cells with and without complementation by the wild type or mutant WRN genes. The graph summarizes three independent experiments with technical triplicates. Red triangles are means, and boxplots mark first and fourth quartiles and the distributions’ medians.