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. Author manuscript; available in PMC: 2022 Nov 4.
Published in final edited form as: Mol Cell. 2021 Sep 30;81(21):4440–4456.e7. doi: 10.1016/j.molcel.2021.09.008

Figure 4. SCAI limits accumulation of nuclear ssDNA during replication stress.

Figure 4.

(A) U2OS cells transfected with the indicated siRNAs for 72 h, then treated with 2 μM Cisp or 1 mM HU for 18 h, harvested and processed for western blotting with the indicated antibodies. ND-no drug. (B) WT U2OS and two independent SCAI null clones (7, 11) were treated with the indicated doses of cisp for 18 h before immunoblotted. (C) U2OS cells were transduced with vector or siRNA resistant FLAG-SCAI. Cells were then treated with indicated siRNAs, with or without 1.5 μM cisp for 18 h before western blotting. (D) WT and SCAI-null U2OS were treated with the indicated siRNAs for 72 h, then treated with vehicle (ND) or 1.5 μM cisp for 18 h before immunoblotting. (E) WT and REV7-null U2OS were transfected with the indicated siRNAs for 72 h, then treated 2 μM cisp for 18 h before immunoblotting. (F) HeLas were transfected with the indicated siRNA for 60 h before treatment with no drug or 0.5 μM Cisp for 6 h. Cells were processed for RPA2 IF. Intensity of nuclear RPA staining from was quantified in 50 cells using ImageJ and plotted. T-tests, **** = p<0.0001. (G) WT or SCAI null U2OS were BrdU labeled and then treated with vehicle (ND) or 1 μM MMC for 6 h and processed for BrdU IF. Average number of BrdU foci per cell was quantified and plotted on the right. T-tests, **** = p<0.0001.