Fig. 4

HER2 expression and PTEN contribute to APOBEC3 activity. a APOBEC3B mRNA expression following silencing of HER2 expression in BT474 cells by RNAi. HER2 levels were depleted by RNAi and, after 72 h, cells were harvested and mRNA extracted. Following cDNA synthesis, APOBEC3B mRNA levels were determined by quantitative PCR; *p < 0.01 (t-test). siNT non-targeting control siRNA. b BT474 cells were treated as in a and, following lysis, western blots were probed with the indicated antibodies. c BT474 and MDA-MB-361 cells were treated as in a and, following lysis, samples were subjected to cytidine deamination assay to determine levels of APOBEC3 activity. d BT474 cells were treated with 10 nM afatinib or 30 nM lapatinib for 24 h followed by mRNA isolation and quantitative PCR to determine APOBEC3B mRNA expression levels; *p < 0.01, ***p < 0.005 (t-test). e BT474 cells were treated as in d and, following lysis, western blots were probed with the indicated antibodies. f BT474 cells were treated as in d and, following lysis, samples were subjected to cytidine-based deamination assay to determine levels of APOBEC3 activity. g MCF10A cells were treated with or without 2 mM hydroxyurea (HU) and exposed to the indicated drugs for 48 h followed by APOBEC3 cytidine deamination assay. h MDA-MB-453 cells were treated with the indicated drugs for 48 h followed by APOBEC3 cytidine deamination assay. i PTEN levels were depleted from MCF7 cells growing on glass coverslips by RNAi. Cells were fixed and stained with 53BP1 and cyclin A antibodies. The fraction of cyclin A-negative cells displaying more than five 53BP1 nuclear foci were scored; *p < 0.05 (t-test). j PTEN levels were depleted from MCF7 cells by RNAi. After 72 h cells were harvested and samples were subjected to cytidine deamination assay to determine APOBEC3 activity. k APOBEC3 activity in response to RAS induction and hydroxyurea (HU) treatment. MCF10A-ER:HRAS V12 cells were induced with tamoxifen (4-hydroxytamoxifen; 4- OHT) in either the presence or absence of hydroxyurea for 48 h, followed by mRNA isolation, cDNA synthesis and quantitative PCR to determine APOBEC3B expression levels. l MCF10A-ER:HRAS V12 cells were treated as in k. Cells were subsequently lysed and subjected to APOBEC3 cytidine deamination assay. LY LY294002, MK MK2206, NT non-targeting, RAPA rapamycin, UNT untreated