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BMI1 and RNF2-deficiency results in DNA replication-associated instabilities.

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posted on 2020-03-06, 18:32 authored by Anthony Sanchez, Angelo de Vivo, Peter Tonzi, Jeonghyeon Kim, Tony T. Huang, Younghoon Kee

A. HU-arrested U2OS cells show delayed cell cycle progression when BMI1 or RNF2 is depleted by siRNA. siTOP2A was used as a positive control (N = 3 biological replicates). B. (Left) Representative images of U2OS cells stained with 53BP1 and Cyclin A following treatment with siRNAs for control (scrambled), BMI1, RNF2 and TOP2A. (Right) quantification of 53BP1 foci in Cyclin A negative cells (N = 100 from 3 biological replicates). (Bottom Right) Western blot analysis to confirm the siRNA-induced knockdowns. C. Parental (WT) or RNF2 KO T80 cells were untreated or transfected with the 3xFLAG-RNF2 plasmid. At 24 hours post-transfection, cells were fixed and analyzed for 53BP1 foci (in Cyclin A-negative cells). Assays were done in triplicates (N = 50 for each condition). D. Schematic for CFS primer binding locations on FRA3B, FRA7H and FRA16D used in ChIP experiments. E. (Top) qPCR quantification of 53BP1 ChIP in T80 wild type and RNF2 KO cells (N = 3 biological replicates; ***P <0.0005, **P <0.005). (Bottom) Western blot confirmation of 53BP1 IP in wild type and RNF2 KO cells. F. qPCR quantification of anti-53BP1 ChIP in T80 cells transfected with either control or RNF2 siRNAs (N = 3 biological replicates; ***P <0.0005, **P <0.005). G. Clonogenic survival assay determines that T80 cells depleted of BMI1, RNF2 or TOP2A by siRNAs are sensitive to treatment with HU. (N = 3 biological replicates). H. Clonogenic survival assay determines that T80 cells depleted of BMI1, RNF2 or TOP2A by siRNA are sensitive to treatment with Aphidicolin (APH) (N = 3 biological replicates). I. (Top) Representative images showing that U2OS cells depleted of BMI1 or RNF2 by siRNAs harbor increased micronuclei. (Bottom) Quantification of the percentage of cells with micronuclei. (N = 50 from 3 biological replicates).