Supporting Information Figure S1 Purity of CLL sample after CD19 positive selection. FACS analysis was performed on PBMCs (PBMC) and purified CLL cells (CLL) using anti-human CD19 and anti-human CD5 antibodies. The purity of the CLL cells was shown as the percentage number on the CD5+CD19+population. (TIF) Figure S2 Hierarchical clustering of miRNAs expressed in CLL, control B cell, and activated B cells. Heatmap of miRNA expression across 38 patient-derived CLL samples, 9 donor B cell samples, and 6 CpG activated donor B cell samples. miRNA expression is hierarchically clustered on the Y-axis and patient-derived CLL samples or control B cell donors are hierarchically clustered on the X-axis. The relative expression of miRNAs is depicted according to the color scale shown on the right. CB#: donor control B sample; CB# act: donor activated B samples by CpG; L#: CLL samples. (TIF) Figure S3 Comparative marker selective view align- ment miRNA expression distinguishes miRNA expres- sion between unactivated donor B cells from CLL cells. Comparative marker selective view alignment distinguishes upregulated miRNA expression in 9 control, unactivated B cell sample (Y-axis, red) and in 38 CLL cell patient samples (X-axis, blue). Score (Y-axis) refers to the t-test score indicating the metric correlating gene expression and phenotype. The calculation of t- test score is: (m1–m2)/ (s1‘2+s2‘2)‘0.5, where m1 is the mean of class 1 and m2 is the mean of class 2, and s1 and s2 are the standard deviation of class 1 and 2. A high score indicates association with the first phenotype (upregulated in control B cells) and a low score indicates association with the second phenotype (upregulated in CLL cells). False Discovery Rate (FDR) is 0.004 for all the miRNAs shown in the diagram. (TIF) Figure S4 miRNA expression profiling in CD5+B cells in comparison to CD5- B cells. B cells were isolated from four donors and six CLL patient samples by CD19+selection. Control B cells were stained with anti-human CD5 and CD19 antibodies, FACS sorted for CD5-CD19+(CD5-) and CD5+CD19+(CD5+) B cells and miRNA expression was analyzed by RT-PCR. Relative expression of miRNAs in CD5- (square), CD5+control B cells (diamond) and CLL samples (triangle) was normalized to RNU44. Each bar indicates the average expression in each group. p value of the analysis calculated by t-test is shown. (TIF) Figure S5 Signature miRNA expression is not signifi- cantly altered in CLL cells before and after CD19 positive selection. Total RNA extracted from PBMC or purified CD19+CLL cells from three CLL patient samples with high white blood cell counts (.506103/mL blood) (CLL) were analyzed by miRNA-specific RT-PCR. The relative expression of miR-26a, miR-29a, miR-29b, miR-150, miR-155, miR-181a, miR-181b, and miR-223 are shown. (TIF) References MicroRNA in Chronic Lymphocytic Leukemia Figure S6 Inhibition of miR-155 expression by MEK and JNK inhibitors. A. Relative expression of miR-155 in CLL cells treated with MEK (PD98059; PD) and JNK (SP600125; SP) inhibitors. B. Relative expression of miR-155 in CpG-activated B cell treated with PD98059 and SP600125 inhibitors. Cells were cultured with DMSO as a control (DMSO). (TIF) Figure S7 miRNA expression distinguishes unstimu- lated B cells from activated B cells. A. anti-IgM was used to activate B cells for 24 or 48 hrs and miRNA-specific RT-PCRs was performed on control B cells before and after activation. B. The same analysis as in (A) by CD40L for 24 hr or 48 hrs. Co- culturing with the 3T3 feeder cells as the control. C. The same analysis as in (A) after LPS, CpG or poly(I:C) activation for 24 hr or 48 hrs. Error bars indicate the standard deviation of duplicates. (TIF) Table S1 Summary of clinical parameters of CLL patients samples used in these studies. (DOC) miRNAs tested in FlexmiR v2 expression Table S2 profiling. (DOC) Table S3 Cell surface marker expression is compared among unactivated donor B cells, anti-IgM-activated donor B cells, and CLL cells. FACS analysis was performed using FITC-labeled different surface marker antibodies. 16 different CLL samples (L#), 5 donor control B cell samples (CB) and 3 activated B cell samples by anti-IgM F(ab’)2 are shown. (DOC) Table S4 miRNAs correlated with ZAP70 and IgVH status. Wilcoxon rank sum tests were used for whether there were any differences on miRNA expression pattern between patients with different clinical features such as (S4A) ZAP70 status, and (S4B) IgVH status. p-value and q-value for each miRNA are shown. N: patient number. (DOC) Table S5 CLL-specific miRNA signatures identified by different groups. (DOC) Methods S1 (DOC) Acknowledgments We thank the CLL Research Consortium Tissue Bank for determination of IgVH and ZAP-70. We thank Etienne Gagnon for the help in the graphic designs. Author Contributions Conceived and designed the experiments: SL CDN. Performed the experiments: SL HFM HZ CDN. Analyzed the data: SL HFM JL LW DN CDN. Contributed reagents/materials/analysis tools: JR KWW JRB. Wrote the paper: SL HFM CDN. 1. Chiorazzi N, Rai KR, Ferrarini M (2005) Chronic lymphocytic leukemia. N Engl J Med 352: 804–815. 2. Rosenwald A, Alizadeh AA, Widhopf G, Simon R, Davis RE, et al. (2001) Relation of gene expression phenotype to immunoglobulin mutation genotype in B cell chronic lymphocytic leukemia. J Exp Med 194: 1639–1647. 3. Klein U, Tu Y, Stolovitzky GA, Mattioli M, Cattoretti G, et al. (2001) Gene expression profiling of B cell chronic lymphocytic leukemia reveals a homogeneous phenotype related to memory B cells. J Exp Med 194: 1625–1638. 4. Stratowa C, Loffler G, Lichter P, Stilgenbauer S, Haberl P, et al. (2001) CDNA microarray gene expression analysis of B-cell chronic lymphocytic leukemia PLoS ONE | www.plosone.org 8 March 2011 | Volume 6 | Issue 3 | e16956