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. 2021 Oct 11;297(5):101301. doi: 10.1016/j.jbc.2021.101301

Figure 1.

Figure 1

Lamin A/C loss results in nuclease-mediated degradation of stalled RFs.A, immunoblots showing depletion of lamin A/C in HEK-293T and MCF-7 cells lentivirally transduced with shRNAs directed against LMNA gene (shLmna) or luciferase gene (shLuc) as control. Similar levels of lamin B1 expression were observed. B, single-molecule replication analysis performed in HEK-293T cells generated in (A). Images show DNA fibers in cells labeled with IdU 20 min + CldU 20 min as detected by fluorescence confocal microscopy. Tract lengths are measured using ImageJ. Graph shows the tract length ratio CldU/IdU in untreated cells (NT), cells treated with HU to stall RFs (4 mM HU for 3 h), and cells treated with HU and the MRE11 nuclease inhibitor Mirin (50 μM). Graph shows each individual value of all measurements in three biological repeats (three independent lamin A/C depletions), with ∼300 forks measured per experiment. The bar indicates the average ± SEM of individual values of all three biological repeats. Note how the CldU/IdU ratio <1 in shLmna cells treated with HU is rescued by Mirin. All DNA fiber assays in this figure were performed with the same labeling scheme as in (B). C, immunoblots show depletion levels of MRE11 nuclease in cells generated in (A) and transiently transfected with siRNA directed against MRE11 (siMRE11) or negative control (siCtrl). Histone 3 is the loading control. Graph shows tract length ratio CldU/IdU in cells NT or treated with HU. Graph represents individual values of two biological repeats (two independent MRE11 depletions), with ∼200 forks measured per experiment. The bar represents average ± SEM of individual values of the two biological repeats. D, tract length ratio CldU/IdU in MCF-7 depleted of lamin A/C and treated with HU ± Mirin as in (B). E, immortalized MEFs from Lmna+/+ and Lmna−/− were treated with HU ± Mirin as in (B) and CldU/IdU ratio calculated. Graph represents individual values, and the bar represents the average ± SEM of two different lines of MEFs. Immunoblots show lack of expression of lamin A/C, but not lamin B1, in MEFs from Lmna−/− mice compared with Lmna+/+. Vinculin and histone 3 are loading controls. F, tract length ratio CldU/IdU in HEK-293T generated in (A) and either NT or exposed to camptothecin (100 nM CPT) or aphidicholin (200 nM APH). Graph shows individual values, and the bar represents average ± SEM of two biological repeats, with ∼200 fibers measured per experiment. G, cells generated in (A) were lentivirally transduced with either a mutant lamin A (LAΔ50 or progerin), wildtype lamin A (LA), or an empty vector (EV) control. Immunoblots show levels of expression of progerin and lamin A. Graph shows CldU/IdU ratio in these cells either untreated (NT) or exposed to HU. Average ± SEM from three biological repeats is represented, with ∼100 fibers measured in each experiment. Individual values of all three biological repeats are combined in the graph. In all figures, ∗ denotes p value of statistical significance (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; and ∗∗∗∗p < 0.0001). CldU, chlorodeoxyuridine; HEK-293T, human embryonic kidney 293T cells; HU, hydroxyurea; IdU, iododeoxyuridine; MEF, mouse embryonic fibroblast; NT, not treated; RF, replication fork.