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Fig. 3 | Genome Biology

Fig. 3

From: DNA replication stress mediates APOBEC3 family mutagenesis in breast cancer

Fig. 3

Induction of replication stress and APOBEC3 activity in breast cancer cell lines. a MCF10A cells were treated with the indicated drugs for 48 h followed by mRNA extraction, cDNA synthesis and quantitative PCR for APOBEC3B and APOBEC3G expression levels. b MCF10A cells were treated as in a followed by western blotting with the indicated antibodies. c MCF10A cells were treated as in a prior to lysis and a cytidine deamination assay for APOBEC3 activity using probe 2. d MCF10A cells were treated as in a followed by fixation and immunofluorescence for Ser139 γH2AX and S4/8 replication protein A phosphorylation (pRPA). Red asterisks indicate treatments inducing APOBEC3B mRNA, protein expression, activity levels and S4/8 RPA phosphorylation. e MCF10A cells were pre-treated with 300 μM exogenous nucleosides followed by incubation with the indicated drugs for an additional 24 h. Following lysis, APOBEC3 activity was measured by a cytidine deamination assay. f Ribonucleotide reductase subunits RRM1, RRM2 and RRM2B were depleted from MCF10A cells by RNA interference and, after 72 h, cells were lysed and subjected to an APOBEC3 cytidine deamination assay. 5FU 5-fluorouracil, MMS methyl methanesulfonate, siNT non-targeting control siRNA

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