The transcriptional program of terminal granulocytic differentiation by Kim Theilgaard-Mönch, Lars Christian Jacobsen, Rehannah Borup, Thomas Rasmussen, Malene Digmann Bjerregaard, Finn Cilius Nielsen, Jack Bernard Cowland, and Niels Borregaard Blood Volume 105(4):1785-1796 February 15, 2005 ©2005 by American Society of Hematology

Characterization of BM populations highly enriched in PMs, MYs, bm-PMNs, and pb-PMNs. Characterization of BM populations highly enriched in PMs, MYs, bm-PMNs, and pb-PMNs. (A, top row) Wright-Giemsa-stained cytospin preparations demonstrate the shape (× 1000 magnification) and the differential count of the purified BM and PB populations (mean ± SD, n = 3), including myeloblasts (MBs), promyelocytes (PMs), myelocytes (MCs), metamyelocytes (MMs), band cells (BCs), and neutrophils from bone marrow (bm-PMNs) and peripheral blood (pb-PMNs). (Bottom row) Two-color flow cytometry analysis was applied to determine the frequency of residual nongranulocytic cells (CD3, CD19, CD14, glycophorin-A, CD56, CD61) in cell populations following purification (mean ± SD, n = 3). (B, top and middle rows) Example of a typical 3-color flow cytometry analysis of BM and PB populations. (Bottom row) The purified populations were subcategorized as MBs, PMs, MCs/MMs, and BCs/PMNs based on cellular size, granularity, and expression profile of the CD15, CD11b, and CD16 surface markers (mean ± SD, n = 3). Kim Theilgaard-Mönch et al. Blood 2005;105:1785-1796 ©2005 by American Society of Hematology

Developmental distance between PM, MY, bm-PMN, and pb-PMN populations. Developmental distance between PM, MY, bm-PMN, and pb-PMN populations. Schematic illustration of terminal granulocytic differentiation. The Pearson correlation coefficients (γ) were calculated by correlating the transcriptomes (all 44 760 probe sets on microarrays) of populations to define a quantitative measure reflecting the developmental distance between the 4 populations. Kim Theilgaard-Mönch et al. Blood 2005;105:1785-1796 ©2005 by American Society of Hematology

Receptor and receptor ligands. Receptor and receptor ligands. Hierarchical clustering of differentially expressed receptors and receptor ligands in PM, MY, and bm-PMN populations annotated to the gene categories of signal transducers and immunity proteins. The mean relative expression levels for genes in BM populations are presented by a color scale, whereby red reflects high expression (3 × SD), white reflects mean expression, and blue reflects low expression (-3 × SD). Kim Theilgaard-Mönch et al. Blood 2005;105:1785-1796 ©2005 by American Society of Hematology

Identification of new GP candidates. Identification of new GP candidates. (Left) Hierarchical clustering of 6700 differentially expressed genes in PM, MY, and bm-PMN populations. The relative expression levels for genes in each BM population (n = 9) are presented by a color scale, whereby red reflects high expression (3 × SD), white reflects mean expression, and blue reflects low expression (-3 × SD). (Right) Lists of genuine GPs and new GP candidates. Genes were defined as GP candidates if they had expression profiles similar to genuine GPs (ie, assigned to cluster 1, 3, 4) and had reported functions critical for immune response. Kim Theilgaard-Mönch et al. Blood 2005;105:1785-1796 ©2005 by American Society of Hematology

Cell cycle-related genes. Cell cycle-related genes. (A) Hierarchical clustering of differentially expressed cell cycle-related genes in PM, MY, and bm-PMN populations. The mean relative expression levels for genes in BM populations are presented by a color scale, whereby red reflects a high expression (3 × SD), white reflects mean expression, and blue reflects low expression level (-3 × SD). (B) Representative flow cytometry analysis of G1, S, G2/M phases in PM, MY, bm-PMN populations. Cells to the left of the G1 population with less than 2n DNA content are undergoing apoptosis. Kim Theilgaard-Mönch et al. Blood 2005;105:1785-1796 ©2005 by American Society of Hematology

Validation of microarray data by real-time RT-PCR, Western blotting, and flow cytometry analysis. Validation of microarray data by real-time RT-PCR, Western blotting, and flow cytometry analysis. (A) Total RNA was purified from BM and PB populations and subjected to real-time RT-PCR and microarray analysis to compare gene expression profiles for selected marker genes of granulocytic differentiation (mean ± SD, n = 3). (B) Comparison of protein and mRNA profiles in BM and PB populations assessed by Western blotting, flow cytometry, and microarray analysis (mean ± SD, n = 3). Kim Theilgaard-Mönch et al. Blood 2005;105:1785-1796 ©2005 by American Society of Hematology

Apoptosis-related genes. Apoptosis-related genes. Hierarchical clustering of differentially expressed apoptosis-related genes in pm, MY, and bm-PMN populations. The mean relative expression levels for genes in BM populations are presented by a color scale, whereby red reflects high expression (3 × SD), white reflects mean expression level, and blue reflects low expression (-3 × SD). Kim Theilgaard-Mönch et al. Blood 2005;105:1785-1796 ©2005 by American Society of Hematology