PCNA-Dependent Cleavage and Degradation of SDE2 Regulates Response to Replication Stress
Fig 5
SDE2 depletion causes replication-associated DNA damage and a defect in replication stress response.
(A) SDE2 knockdown leads to increased levels of γH2AX upon DNA damage. HeLa cells transfected with the indicated siRNAs were treated with 40 J/m2 UVC for 4 h and analyzed by Western blotting. (B) (left) siRNA-transfected U2OS cells were treated with 40 J/m2 UVC for 4 h, and γH2AX foci were analyzed by immunofluorescence. (right) Quantification of cells displaying more than 10 γH2AX foci. Data shown are the mean ± SD from three independent experiments. * p < 0.01 compared with siRNA control. (C) MUS81 depletion suppresses damage-induced γH2AX caused by SDE2 knockdown. HeLa cells transfected with indicated siRNA oligoes were treated with 40 J/m2 for 4 h, and cell lysates were analyzed by Western blotting. Note that PCNA monoubiquitination was decreased upon MUS81 knockdown (related to Fig 7). (D, E) Luminescence-based viability (D) or clonogenic survival (E) of siRNA-transfected HeLa cells treated with the indicated doses of DNA damage. Data shown are the mean ± SD from three independent experiments. * p < 0.01 SDE2 knockdown compared with control (except 250 μM HU p < 0.05). (F) SDE2 knockdown causes a defect in S phase progression. HeLa cells transfected with siRNA control or SDE2 were synchronized at G2/M phase by treating 100 ng/mL nocodazole for 16 h. After mitotic shake-off, cells were released into G1 and S phases, and cell cycle was monitored by PI staining and flow cytometry. Data shown are the mean ± SD from three independent experiments. * p < 0.05 for S phase population from cells with SDE2 knockdown vs. control. (G) HeLa cells transfected with siRNA control or SDE2 were left untreated or treated with 40 J/m2 UVC, and incubated with 10 μM BrdU for 0.5 h before harvest at 4 h post UVC irradiation. S phase cells were determined by anti-BrdU/PI staining and flow cytometry, and SDE2-depleted BrdU+ cells were normalized by control-treated BrdU+ cells. Data shown are the mean ± SD from two independent experiments. * p < 0.01 SDE2 knockdown vs. control. (H) Decreased replication recovery of SDE2-depleted cells against UV damage. HeLa cells transfected with siRNA control or SDE2 were pulsed with 10 μM BrdU for 0.5 h, left untreated or treated with 40 J/m2 UVC, and released into fresh medium for 4 h. (left) Representative cell cycle distribution measured by anti-BrdU/PI staining and flow cytometry. (right) Relative distribution of early S (A/A+B) and late S (B/A+B) cells out of total BrdU+ cells. Data shown are the mean ± SD from three independent experiments. * p < 0.01 for increased early and decreased late S populations from cells with SDE2 knockdown vs. control.