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

Figure 3.

Figure 3

Replication defects in lamin A/C-deficient cells have similarities with BRCA2-deficient cells.A, HEK-293T cells depleted of lamin A/C (shLmna) or control cells (shLuc) were lentivirally transduced with short hairpin directed against BRCA2 or a scrambled control (shscr). Immunoblots show reduced levels of BRCA2 and lamin A/C proteins. Vinculin serves as loading control. B, single-molecule replication analysis performed in HEK-293T cells generated in (A). Cells were labeled with IdU 20 min + CldU 20 min and left untreated or exposed to 4 mM HU for 5 h. Graph shows average ± SEM of tract length ratio CldU/IdU in cells generated in (A) and either left untreated (NT) or treated with HU. Cells depleted of BRCA2, lamin A/C, or both proteins share similar phenotypes under replication stress (CldU/IdU < 1). Data from two independent experiments from two biological repeats, with ∼200 fibers measured per experiment. Graph shows each individual value of all measurements from the two biological repeats. C, cells generated in (A) and labeled as in (B) are exposed to HU alone or in combination with Mirin and used in single-molecule replication analysis. Graph shows tract length ratio CldU/IdU. Note how inhibition of MRE11 rescues fork degradation similarly in all the three different lines. Data from two independent experiments from two biological repeats, with ∼200 fibers measured per experiment. Graph shows each individual value from the two biological repeats, and the bar indicates the average ± SEM. D, immunoblots showing siRNA-mediated depletion of RAD51 (left panels) and BRCA2 and RAD51 (right panels) in lamin A/C-proficient and lamin A/C-deficient cells. Vinculin is the loading control. Representative image of three biological repeats. E, graph shows tract length ratio CldU/IdU of cells generated in (D) and labeled with IdU 20 min + CldU 20 min prior to exposure to HU for 5 h. Note how RAD51 depletion rescues RFI in BRCA2-deficient cells but not in lamin A-deficient cells. Graph shows each individual value of all measurements from all three replicates. The bar indicates the average of all measurements ± SEM. F, immunoblots showing lamin A/C-proficient and lamin A/C-deficient cells transfected with siRNAs for depletion of BRCA2 and SMARCAL1. Graph shows tract length ratio CldU/IdU of lamin A/C-proficient and lamin A/C-deficient cells depleted of SMARCAL1 and/or BRCA2 via siRNAs. Note how SMARCAL1 depletion rescues resection in BRCA2-deficient cells but not in lamin A/C-deficient cells. Graph shows each individual value of all measurements from two replicates. The bar indicates the average of all measurements ± SEM. G, cell lines generated in (A) were transduced with RAD51 construct or an empty vector (EV) control. Immunoblots show the overexpression of RAD51 in the three cell lines. Graph shows the CldU/IdU ratio in the six different cells. Note how overexpression of RAD51 rescues replication defects in all three lines. Data from three biological repeats and ∼200 fibers measured in each experiment. Graph shows each individual value of all measurements from all three replicates. The bar indicates the average of all individual measurements ± SEM. BRCA, BReast CAncer gene; CldU, chlorodeoxyuridine; dU, iododeoxyuridine; HEK-293T, human embryonic kidney 293T cells; HU, hydroxyurea; NT, not treated; RFI, replication fork instability.