Extended Data Fig. 6: Validation of variability observed with antibody staining.
From: Spatiotemporal dissection of the cell cycle with single-cell proteogenomics
a, Box plot showing gene expression levels for all proteins that exhibit cell-to-cell heterogeneity and all proteins in the HPA Cell Atlas (mapped proteome). There is no significant difference (P < 0.01) in gene expression levels between the antibodies included in this study and all antibodies used in the HPA Cell Atlas (P = 0.03, two-sample Student’s t-test; variation, n = 1,607; mapped proteome, n = 9,806 independent proteins). b, Bar plot showing staining intensity levels for antibodies included in this study and all proteins in the HPA Cell Atlas (mapped proteome). The average immunofluorescence signal intensity measured for proteins showing cell-to-cell variation and for all proteins in the HPA Cell Atlas shows no significant difference for low signal antibodies. Low signal antibodies are not enriched among the cell-to-cell variability dataset (strong, P = 1.12 × 10−5; moderate, P = 0.0004; weak, P = 0.99 by binomial one-sided tests; asterisk denotes significance). c, Since the HPA antibodies are purified on affinity columns coupled with their corresponding antigen, antibody concentration after purification can serve as a proxy for antibody affinity (albeit not a perfect proxy). Box plot showing antibody concentration levels for all proteins that showed cell-to-cell heterogeneity and all proteins in the HPA Cell Atlas (mapped proteome). There is no significant difference between the antibodies included in this study and all antibodies used in the HPA Cell Atlas, hence we can conclude that the cell-to-cell heterogeneity is probably not due to differences in antibody affinity. The average antibody concentration for the antibodies published on the HPA Cell Atlas is 0.1710 mg ml−1, and the average concentration for the antibodies used in this study is 0.1712 mg ml−1 (P = 0.1084, two-sample Student’s t-test; variation, n = 1,415; mapped proteome, n = 14,942 independent proteins). d, Box plot showing the variance for all proteins that showed cell-to-cell heterogeneity and microtubules. Proteins showing heterogeneity show significantly higher variance than the variance of the microtubules measured from each well (P = 1.6 × 10−292, one-sided Kruskal–Wallis test; n = 1,180 independent IF stains). e, Box plot showing the Gini index for all proteins that showed cell-to-cell heterogeneity and for microtubules. Proteins displaying heterogeneity show significantly higher Gini indexes than microtubules (P < 5 × 10−324, one-sided Kruskal–Wallis test; n = 1,180 independent IF stains). f, Gene ontology enrichment analysis for CCD proteins shows significantly enriched terms for the biological processes domain. Each circle represents a GO term, and line width corresponds to the number of genes that overlap between the two connected gene sets. Similar terms are grouped and labelled. g, GO enrichment analysis for non-CCD enzymes shows enrichment for basic metabolic functions, while CCD enzymes are enriched for cell cycle functions. h, Subcellular localizations of CCD and non-CCD proteins. Asterisk denotes enrichment relative to the HPA-mapped proteome. For CCD proteins: cleavage furrow, P = 0.0003; cytokinetic bridge, P = 4.3 × 10−93; kinetochores, P = 3.5 × 10−5; midbody ring, P = 2.3 × 10−15; midbody, P = 9 × 10−36; mitotic chromosome, P = 9.8 × 10−66; mitotic spindle, P = 6.3 × 10−41; nucleoli, P = 0.005. For non-CCD proteins: intermediate filaments, P = 1.9 × 10−8; mitochondria, P = 1.9 × 10−11; nuclear bodies, P = 3.01 × 10−5; nucleoli, P = 8.8 × 10−7. i, Protein–protein interaction (PPI) network of CCD proteins and CCD transcripts using the STRING70 database. Proteins with known associations to the cell cycle (by GO term, teactome pathway, or cyclebase phenotype) are represented as green squares (left). Transcriptionally regulated CCD proteins are tightly clustered in the centre of the network, with an extended network of novel CCD genes and particularly ones that are not transcriptionally regulated (right). For box plots: centre line, median; box, Q1 and Q3; whiskers, 1.5× IQR below Q1 and above Q3; points, outliers.
