A Cell-Line-Specific Atlas of PARP-Mediated Protein Asp/Glu-ADP-Ribosylation in Breast Cancer  Yuanli Zhen, Yajie Zhang, Yonghao Yu  Cell Reports  Volume 21, Issue 8, Pages 2326-2337 (November 2017) DOI: 10.1016/j.celrep.2017.10.106 Copyright © 2017 The Author(s) Terms and Conditions

Cell Reports 2017 21, 2326-2337DOI: (10.1016/j.celrep.2017.10.106) Copyright © 2017 The Author(s) Terms and Conditions

Figure 1 General Scheme of Profiling Cell-Specific D/E-ADP-Ribosylated Proteins (A) Flowchart of global characterization of the D/E-ADP-ribosylated proteome in breast (cancer) cells. (B) Breast (cancer) cells display profound variation in their overall PARylation levels. All cell lines were treated with 2 mM H2O2 for 5 min. Lysates were probed with an anti-PAR antibody and the indicated antibodies. (C) The number of identified ADP-ribosylated peptides (blue) and ADP-ribosylated proteins (red) for each cell line. (D–G) Gene ontology (GO) analysis for the PARylated protein identified from MCF10A (D), MCF7 (E), SK-BR-3 (F), and MDA-MB-468 (G) cells. (H) The PARylated proteins commonly identified among ER+ cell lines (MCF7, T47D, and ZR-75-1) and among cell lines from different breast cancer subtypes (MCF7, SK-BR-3, and MDA-MB-468). (I) Hierarchical clustering of the PARylated proteins and cell lines based on total spectral counts (TSC). The total number of PARylated peptides identified for each protein was summed (TSC) in each cell line, which was used to generate this heatmap. Cell Reports 2017 21, 2326-2337DOI: (10.1016/j.celrep.2017.10.106) Copyright © 2017 The Author(s) Terms and Conditions

Figure 2 General Scheme of Protein Expression Profiling of the Breast (Cancer) Cell Line Panel (A) Flowchart of the TMT experiments for global protein expression analyses. (B) The TMT labeling scheme and a summary of the quantification results. Two TMT sets were performed to quantify the protein expression in the 9 breast cell lines. Only data from the first biological replicate TMT experiment were listed. (C) Reproducibility of the two biological replicate TMT experiments. Raw S/N (signal-to-noise ratio) values of each protein in T47D cells were extracted from the two biological replicate TMT samples and were plotted. (D) PCA (principal-component analysis) of the breast (cancer) cell lines based on their protein expression. (E) Hierarchical clustering of protein expression and cell lines (normalized to MCF10A, shown as log2-transformed ratio). Representative proteins that are overexpressed in cancer cell lines were subject to GO analysis. (F) Comparison of protein expression between MCF7 cells and MCF10A cells. The abundance of each protein in these two cell lines was extracted, and the ratio (MCF7/MCF10A) was plotted on a log2 plot. Proteins that are upregulated (more than 10-fold) or downregulated (more than 10-fold) in MCF7 cells are highlighted and are subject to GO analysis. (G–I) Representative upregulated proteins (normalized to MCF10A, shown as ratio) in ER+ cell lines (G), TNBC lines (H), and HCC1937 cells (I). Cell Reports 2017 21, 2326-2337DOI: (10.1016/j.celrep.2017.10.106) Copyright © 2017 The Author(s) Terms and Conditions

Figure 3 Cross-Cell Line Comparison of Protein Expression and ADP-Ribosylation (A) Clustering of proteins based on their ADP-ribosylation TSC values (upper diagram; normalized to MCF10A, shown as ratio) and protein expression (lower diagram; normalized to MCF10A, shown as ratio). These two datasets were then cross-referenced to determine the Spearman’s rank correlation coefficient (ρ) for each protein. Proteins were ranked based on their corresponding ρ values. (B) Total PARylated peptides versus PARP1 PARylated peptides identified in each cell line (normalized to those in MCF10A cells). (C) GATA3 ADP-ribosylated peptides (TSCs) identified in each cell line (normalized to those in MCF10A cells). Also shown is an immunoblotting analysis of GATA3 in these cells. (D) THOC4 ADP-ribosylated peptides (TSCs) identified in each cell line (normalized to those in MCF10A cells). Also shown is an immunoblotting analysis of THOC4 in these cells. (E) Heatmap depicting TSCs of the various THOC4 ADP-ribosylation sites in the cell lines. (F) THOC4 domain structure depicting representative breast-cancer-specific ADP-ribosylation sites versus ADP-ribosylation sites identified in all cell lines. (G and H) MS2 spectra of representative ADP-ribosylated peptides identified from GATA3 in T47D (G) and THOC4 in MDA-MB-468 (H), respectively. The asterisk indicates the ADP-ribosylation site. Cell Reports 2017 21, 2326-2337DOI: (10.1016/j.celrep.2017.10.106) Copyright © 2017 The Author(s) Terms and Conditions

Figure 4 PARylated Proteins in MDA-MB-468 Cells Are Enriched with Those Involved in Protein Translation (A–C) Mapping ADP-ribosylated proteins onto protein interaction networks using the STRING database. Representative functional clusters are shown for MDA-MB-468 (A), MDA-MB-231 (B), and MCF7 (C) cells. (D) Ribosomal proteins were selectively ADP-ribosylated in MDA-MB-468 cells. The heatmap is generated by plotting the TSC of each PARylated protein for the corresponding cell lines. (E) Topology of the PARylated ribosomal proteins within the 80S ribosome. The cryo-EM structure of 80S ribosome was downloaded from the PDB (4UG0), with the identified PARylated proteins highlighted in red. One of these modified proteins RPL8 (uL2) is indicated by an arrow. (F) Illustration of the inter-subunit bridge between RPS6 (eS6) (green) and RPL24 (eL24) (red). (G) Illustration of the inter-subunit bridge between 28S rRNA (yellow) and RPS8 (eS8) (red). Cell Reports 2017 21, 2326-2337DOI: (10.1016/j.celrep.2017.10.106) Copyright © 2017 The Author(s) Terms and Conditions