Research Paper Volume 16, Issue 20 pp 12977—13011

Figure 11. Lamin B1 association with replicating DNA is reduced in WRN-deficient cells. (A) An example of a PLA analysis detecting Lamin B1 association with nascent DNA in situ, demonstrating specificity of the PLA signal to EdU+ positive cells. WI38hTERT cells (expressing non-targeting shRNA) pulse-labeled with 20 µM EdU for 10 min and collected after a 10 min chase. EdU was clicked to a mixture of biotin-azide and Alexa488-azide at a molar ratio of 50:1. PLA was performed with antibodies against Lamin B1 and biotin. Scale bar, 10 µm. (B) Quantitation of EdU incorporation in WI38hTERT expressing the indicated shRNAs. (C) Quantitation of LMNB1/EdU PLA in the same experiment as in (B). The datasets were subsetted into EdU- and EdU+ groups by applying a fixed cut-off EdU MFI value (e.g., 0.02 in (B)), below which cells were deemed EdU-. Lamin B1/EdU PLA foci counts were then plotted separately for EdU- and EdU+ subsets. (D) For each cell in EdU+ subpopulations, Lamin B1/EdU PLA foci counts were normalized to EdU MFI values and plotted. This normalization compensates for a somewhat lower level of EdU incorporation typically seen in WRN-depleted cells compared to the control (B). P-values were calculated in Wilcoxon tests. Blue crossbars are medians and red crossbars are means. The results of (B–D) represent two independent experiments, one of which included biological triplicates. (E) A model of WRN roles in CH maintenance. (1) WRN is proximal to HDAC2 via HP1α in the context of Lamin B1/LBR-tethered CH assemblies at the inner nuclear membrane (INM). Reduction of LBR and Lamin B1 in the absence of WRN partially disrupts these assemblies. WRN supports LBR enrichment within the nucleus (green arrow). Possible mechanisms include action on regulators of LBR localization or on nuclear pore complexes. (2) WRN prevents accumulation of R-loops and/or G-quadruplexes in the introns of the LBR gene using its canonical enzymatic activities. Suppression of R-loops and/or G-quadruplexes may promote proper splicing of the LBR transcript.