Journal: PLoS Genetics

Article Title: APOBEC3A is a prominent cytidine deaminase in breast cancer

doi: 10.1371/journal.pgen.1008545

Figure Lengend Snippet: (A) The mutation profile of AU565 and SKBR3 BRCA cell lines. (B) mRNA expression level of individual APOBEC3 family members relative to HPRT1 expression in AU565 (black) and SKBR3 (grey). Bars indicate the mean values of 3 replicate measurements. Error bars indicate the standard error of the mean (SEM) of these measurements. n.d. indicates “not detected.” Similar results were obtained using TBP instead of HPRT1 as the internal reference gene . (C) Schematic of in vitro cytidine deaminase assay. (D) AU565, AU565 cells containing a CRISPR-Cas9 mediated disruption of APOBEC3A (-/-), and (E) SKBR3 BRCA cell lines either un-transduced or expressing scramble control, A3A shRNA, or A3B shRNA were tested for cytidine deaminase activity on a hairpin or linear substrate containing a YTCA APOBEC target motif. Each cell line was additionally transduced to express a vector control or uracil glycosylase inhibitor (UGI) as indicated. 40 μg of total protein was incubated with 0.25 μM of hairpin substrate for 24 hrs at 37°C, prior to heating the samples at 95°C for 10 min and separating substrate from cleavage product on a denaturing polyacrylamide gel. Knockdown specificity was confirmed by qRT-PCR and equal protein amount in each reaction was confirmed via α-GAPDH western.

Article Snippet: RSEM RNA-seq expression values for BRCA cell lines and TCGA sequenced primary tumors were obtained from the Cancer Cell Line Encyclopedia ( https://data.broadinstitute.org/ccle/CCLE_RNAseq_rsem_transcripts_tpm_20180929.txt.gz ) and the Broad Institute Genomic Data Analysis Center ( http://gdac.broadinstitute.org/runs/stddata__2016_01_28/data/BRCA/20160128/gdac.broadinstitute.org_BRCA.Merge_rnaseqv2__illuminahiseq_rnaseqv2__unc_edu__Level_3__RSEM_genes_normalized__data.Level_3.2016012800.0.0.tar.gz ; http://gdac.broadinstitute.org/runs/stddata__2016_01_28/data/BLCA/20160128/gdac.broadinstitute.org_BLCA.Merge_rnaseqv2__illuminahiseq_rnaseqv2__unc_edu__Level_3__RSEM_genes_normalized__data.Level_3.2016012800.0.0.tar.gz ; http://gdac.broadinstitute.org/runs/stddata__2016_01_28/data/CESC/20160128/gdac.broadinstitute.org_CESC.Merge_rnaseqv2__illuminahiseq_rnaseqv2__unc_edu__Level_3__RSEM_genes__data.Level_3.2016012800.0.0.tar.gz ; and http://gdac.broadinstitute.org/runs/stddata__2016_01_28/data/HNSC/20160128/gdac.broadinstitute.org_HNSC.Merge_rnaseqv2__illuminahiseq_rnaseqv2__unc_edu__Level_3__RSEM_genes_normalized__data.Level_3.2016012800.0.0.tar.gz ), respectively.

Techniques: Mutagenesis, Expressing, In Vitro, CRISPR, Disruption, Control, shRNA, Activity Assay, Plasmid Preparation, Incubation, Knockdown, Quantitative RT-PCR, Western Blot

Journal: PLoS Genetics

Article Title: APOBEC3A is a prominent cytidine deaminase in breast cancer

doi: 10.1371/journal.pgen.1008545

Figure Lengend Snippet: The average mutation profiles of (A) 14 non-APOBEC-mutated and (B) 14 APOBEC-mutated BRCA cell lines. Specific cell lines in each category are defined in . (C) mRNA expression level of individual APOBEC3 family members relative to HPRT1 expression was measured by qRT-PCR in non-APOBEC-mutated (N) and APOBEC-mutated (M) BRCA cell lines. Similar results were obtained comparing APOBEC expression to TBP . Each circle represents the mean of 3 replicate measurements for an individual cell line. Horizontal bars indicate the median expression for each APOBEC3 family member among the non-APOBEC-mutated or APOBEC-mutated cell lines. Data points corresponding to cell lines without detectable expression of individual APOBECs are not shown on the graph but are included in the calculation of the median. Statistical significance for differences in the expression of a given APOBEC family member between non-APOBEC-mutated and APOBEC-mutated lines was assessed by Mann-Whitney Summed Rank test. ** indicates p = 0.0067. Correlations between A3A expression (blue dots) or A3B expression (red dots) measured by qRT-PCR and the minimum estimate of APOBEC-induced mutations for each of the 28 BRCA cell lines were determined by a Pearson correlation test using mutation lists obtained from (D) the Cancer Cell Line Encyclopedia and (E) the Catalogue of Somatic Mutations in Cancer (COSMIC).

Article Snippet: RSEM RNA-seq expression values for BRCA cell lines and TCGA sequenced primary tumors were obtained from the Cancer Cell Line Encyclopedia ( https://data.broadinstitute.org/ccle/CCLE_RNAseq_rsem_transcripts_tpm_20180929.txt.gz ) and the Broad Institute Genomic Data Analysis Center ( http://gdac.broadinstitute.org/runs/stddata__2016_01_28/data/BRCA/20160128/gdac.broadinstitute.org_BRCA.Merge_rnaseqv2__illuminahiseq_rnaseqv2__unc_edu__Level_3__RSEM_genes_normalized__data.Level_3.2016012800.0.0.tar.gz ; http://gdac.broadinstitute.org/runs/stddata__2016_01_28/data/BLCA/20160128/gdac.broadinstitute.org_BLCA.Merge_rnaseqv2__illuminahiseq_rnaseqv2__unc_edu__Level_3__RSEM_genes_normalized__data.Level_3.2016012800.0.0.tar.gz ; http://gdac.broadinstitute.org/runs/stddata__2016_01_28/data/CESC/20160128/gdac.broadinstitute.org_CESC.Merge_rnaseqv2__illuminahiseq_rnaseqv2__unc_edu__Level_3__RSEM_genes__data.Level_3.2016012800.0.0.tar.gz ; and http://gdac.broadinstitute.org/runs/stddata__2016_01_28/data/HNSC/20160128/gdac.broadinstitute.org_HNSC.Merge_rnaseqv2__illuminahiseq_rnaseqv2__unc_edu__Level_3__RSEM_genes_normalized__data.Level_3.2016012800.0.0.tar.gz ), respectively.

Techniques: Mutagenesis, Expressing, Quantitative RT-PCR, MANN-WHITNEY

Journal: PLoS Genetics

Article Title: APOBEC3A is a prominent cytidine deaminase in breast cancer

doi: 10.1371/journal.pgen.1008545

Figure Lengend Snippet: (A) The pairwise alignment of the A3A (blue) and A3B (red) transcripts shows a central region of high sequence identity with two unique regions in each transcript. Green, yellow, and red indicate 100%, ≥30%, and <30% identity respectively. Arrows indicate the position of qRT-PCR primers. (B) Correlations between qRT-PCR measurements of A3A or A3B mRNA abundance and corresponding RSEM normalized RNA-seq measurements or RNA-seq measurements limited to reads mapping within the defined unique regions of A3A and A3B transcripts (unambiguous RNA-seq) for BRCA cell lines were assessed by Pearson correlation test. A3A and A3B expression measured by RSEM normalized RNA-seq or unambiguous RNA-seq was compared to the minimum estimate of APOBEC-induced mutations in (C) 973 TCGA sequenced primary BRCA tumors or (D) a subset of 229 APOBEC-mutated TCGA sequenced primary BRCA tumors by Pearson correlation test.

Article Snippet: RSEM RNA-seq expression values for BRCA cell lines and TCGA sequenced primary tumors were obtained from the Cancer Cell Line Encyclopedia ( https://data.broadinstitute.org/ccle/CCLE_RNAseq_rsem_transcripts_tpm_20180929.txt.gz ) and the Broad Institute Genomic Data Analysis Center ( http://gdac.broadinstitute.org/runs/stddata__2016_01_28/data/BRCA/20160128/gdac.broadinstitute.org_BRCA.Merge_rnaseqv2__illuminahiseq_rnaseqv2__unc_edu__Level_3__RSEM_genes_normalized__data.Level_3.2016012800.0.0.tar.gz ; http://gdac.broadinstitute.org/runs/stddata__2016_01_28/data/BLCA/20160128/gdac.broadinstitute.org_BLCA.Merge_rnaseqv2__illuminahiseq_rnaseqv2__unc_edu__Level_3__RSEM_genes_normalized__data.Level_3.2016012800.0.0.tar.gz ; http://gdac.broadinstitute.org/runs/stddata__2016_01_28/data/CESC/20160128/gdac.broadinstitute.org_CESC.Merge_rnaseqv2__illuminahiseq_rnaseqv2__unc_edu__Level_3__RSEM_genes__data.Level_3.2016012800.0.0.tar.gz ; and http://gdac.broadinstitute.org/runs/stddata__2016_01_28/data/HNSC/20160128/gdac.broadinstitute.org_HNSC.Merge_rnaseqv2__illuminahiseq_rnaseqv2__unc_edu__Level_3__RSEM_genes_normalized__data.Level_3.2016012800.0.0.tar.gz ), respectively.

Techniques: Sequencing, Quantitative RT-PCR, RNA Sequencing, Expressing