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. Author manuscript; available in PMC: 2014 Dec 12.
Published in final edited form as: Mol Cell. 2013 Nov 14;52(5):10.1016/j.molcel.2013.10.012. doi: 10.1016/j.molcel.2013.10.012

Figure 3. TopBP1 Suppresses SCE by Maintaining BLM Expression.

Figure 3

(A) Depletion of TopBP1 decreases BLM protein level. HeLa cells transfected with control (CTR) or TopBP1 siRNA were mock treated or treated with hydroxyurea (HU, 10 mM). Cells were harvested and immunoblotted with the indicated antibodies.

(B) BLM is not required for Chk1 activation in response to replication stress. Wild-type and BLM-deficient cells were mock treated or treated with HU (10 mM). Cell lysates were immunoblotted with antibodies as indicated.

(C) Depletion of TopBP1 increases SCE frequency. HeLa cells were transfected with CTR, TopBP1, or ATR siRNAs. SCE assay were performed. Numbers of SCEs per chromosome were evaluated under the microscope. The results were the average of three independent experiments and presented as mean ± SD (left panel). Western blotting was carried out to verify the knockdown of ATR and BLM protein level in ATR knockdown cells (right panel).

(D) BLM overexpression suppressed SCE in TopBP1-depleted cells. HeLa cells were transfected with CTR or TopBP1 siRNAs twice in a 24-hr interval with or without co-transfection with constructs encoding wild-type, SFB-tagged BLM. Numbers of SCEs per chromosome were evaluated under the microscope in these transfected cells. The results were the average of three independent experiments and presented as mean ± SD (left panel). Western blotting was carried out to verify the overexpression of wild-type SFB-BLM (right panel).

(E) U2OS cells stably expressing siRNA-resistant constructs encoding wild-type, ΔBRCT5 mutant, or ΔAD mutant SFB-TopBP1 were transfected with CTR siRNA or TopBP1 specific siRNA. Numbers of SCEs per chromosome were evaluated under the microscope. The results were the average of three independent experiments and presented as mean ± SD (left panel). Western blotting was carried out to confirm the expression of wild-type or mutant TopBP1 in these cells (right panel).