made the measurements from day-to-day without knowing the treatment regimen the mice received. At day 21, host mice were euthanized, the lymphomas were excised, and single cell suspensions were made of each to determine the fraction of tumor cells expressing GFP. Cell Cycle and Apoptotic Analysis The culture of GFP+ A20 cells was maintained as described above. At each time-point, cell cycle analysis was measured by BrdU labeling using BrdU Flow Kit (Pharmingen, San Diego, CA) according to the manufacturer’s instruction. 10 ul of BrdU solution (1 mM) was added to 16106 cells in 1 ml culture medium and incubated for 1 hour. The cells were fixed and permeabilized by Cytofix/Cytoperm Buffer and treated with 30 ug DNase for 1 hour at 37uC. After washing with Perm/Wash buffer, cells were stained with PE-conjugated anti-BrdU antibody for 20 minutes at room temperature, washed and 20 ul of 7-AAD was added. The cells were analyzed by flow cytometry on Facscan (Becton Dickinson Immunocytometry Systems, San Jose, CA). Immunohistochemstry of A20 Cells Treated with Potato Carboxypeptidase Inhibitor (PCPI) PCI was purchased from Sigma-Aldrich Co. (St. Louis, MO). FITC labeling of PCI was performed by using FLUOROTAGTM FITC CONJUGATION KIT (Sigma-Aldrich) according to manufacturer’s instruction, and was used to treat A20 cells for fluorescence internalization assays. Cells are cultured on 22622 mm microscope cover glasses (Fisher Scientific Co., Pittsburgh, PA) in media as described above. Cells were fixed onto cover glasses with 1:1 methanol:acetic acid and washed 3 times by PBS. Cells were incubated with FITC-conjugated PCI at a concentration of 30 ug/ml at 37uC for 30 minutes, washed and stained with phycoerythrin (PE)-conjugated B220 and DAPI. The cover glass coated with A20 cell monolayer was flipped immediately and sealed onto glass slide. The image was taken with a Zeiss Axiovert- 200 microscope using a high-resolution Zeiss digital camera (Carl Zeiss Inc., Thornwood, NY). Culture of A20 Cells with Potato Carboxypeptidase Inhibitor To determine the effects of PCPI on A20 cell growth, A20 cells were seeded at a density of 66105/well in 6-well plates, cultured overnight before the addition of 0, 5, 15, 30 or 60 ug/ml PCI. The cells were fed every 2 days with fresh medium (as above) containing the respective concentration of PCI, and viable cells were counted on a hemacytometer using trypan blue dye exclusion. Cells were split according to cell population size to maintain a cell concentration of 2–56106/ml and cultures were maintained for 12 days. The cumulative cell number was References 1. Dick JE, Lapidot T (2005) Biology of normal and acute myeloid leukemia stem cells. Int J Hematol 82: 389–396. 2. Marx J (2007) Molecular biology. Cancer’s perpetual source? Science 317: 1029–1031. 3. Huntly BJ, Gilliland DG (2005) Leukaemia stem cells and the evolution of cancer-stem-cell research. Nat Rev Cancer 5: 311–321. 4. Liang Y, Jansen M, Aronow B, Geiger H, Van Zant G (2007) The quantitative trait gene latexin influences the size of the hematopoietic stem cell population in mice. Nat Genet 39: 178–188. 5. Arimatsu Y (1994) Latexin: a molecular marker for regional specification in the neocortex. Neurosci Res 20: 131–135. 6. Hatanaka Y, Uratani Y, Takiguchi-Hayashi K, Omori A, Sato K, et al. (1994) Intracortical regionality represented by specific transcription for a novel protein, latexin. Eur J Neurosci 6: 973–982. Latexin: A Potential Tumor Suppressor calculated from the cell counts and the dilutions made at each culture split. Statistical Analysis Data were analyzed by either student t-test assuming unequal variance with P,0.05 (two-tail), or a one-way ANOVA. Supporting Information Figure S1 Cpa3 is not expressed in A20 cells. Real-time PCR was performed on A20 cells that were either uninfected (A20 control) or infected with empty (vector control) or Lxn expression vector (Lxn vector) to quantify Cpa3 mRNA expression. The amplification plots for Lxn (A), Cpa3 (B) and Gapdh (C) transcript show that Lxn is highly expressed in A20 cells infected with Lxn expression vector whereas A20 and vector controls have very low expression levels, consistent with the results in Fig. 3c. Cpa3 transcript is not amplified in all types of cells, indicating it is not expressed in A20 cells. These figures show the amplification plots of four individual biological replicates (n = 4) for each gene. (TIF) Table S1 Carboxypeptidase A3 (Cpa3) is highly ex- pressed in stem/progenitor cells. Expression level of CPAs were measured by microarray on a bone marrow population for cell markers characteristic of lineage-specific differentiated blood cells (Lin-negative), and positive for the Sca-1 and c-Kit cell markers (LSK) cells, enriched for hematopoietic stem/progenitor cells in mouse. The isolation of LSK cells as well as microarray analysis was performed as previously described [38] except the array platform is mouse Genome 430 2.0 Array (Affymetrix). Three biological samples were assayed, and the mean expression value (mean_y_B6) for each gene in the table is the average of three readings. Cpa3 (highlighted in gray) is the only Cpa gene that is highly enriched in LSK cells. Gapdh is the endogenous control. (PDF) Acknowledgments We gratefully acknowledge Dr. Gary Van Zant for reviewing the manuscript. We thank the technical assistance of Dr. Xian Gao in image capturing of immunocytochemstry staining, Barry Grimes in flow cytometry, and Hope Johnson and Paula Thomason for editing manuscript. Author Contributions Conceived and designed the experiments: Y. Liang. Performed the experiments: Y. Liu KR CS Y. Liang. Analyzed the data: Y. Liu Y. Liang. Contributed reagents/materials/analysis tools: JB LS JM JSB SB DH. Wrote the paper: Y. Liang. 7. Arimatsu Y, Ishida M, Takiguchi-Hayashi K, Uratani Y (1999) Cerebral cortical specification by early potential restriction of progenitor cells and later phenotype control of postmitotic neurons. Development 126: 629–638. 8. Arimatsu Y, Kojima M, Ishida M (1999) Area- and lamina-specific organization of a neuronal subpopulation defined by expression of latexin in the rat cerebral cortex. Neuroscience 88: 93–105. 9. Arimatsu Y, Ishida M, Kaneko T, Ichinose S, Omori A (2003) Organization and development of corticocortical associative neurons expressing the orphan nuclear receptor Nurr1. J Comp Neurol 466: 180–196. 10. Jin M, Ishida M, Katoh-Fukui Y, Tsuchiya R, Higashinakagawa T, et al. (2006) Reduced pain sensitivity in mice lacking latexin, an inhibitor of metallocarbox- ypeptidases. Brain Res 1075: 117–121. 11. Aagaard A, Listwan P, Cowieson N, Huber T, Ravasi T, et al. (2005) An inflammatory role for the mammalian carboxypeptidase inhibitor latexin: PLOS ONE | www.plosone.org 11 September 2012 | Volume 7 | Issue 9 | e44979