Latexin: A Potential Tumor Suppressor Figure 4. Ectopic Lxn expression increases apoptosis of A20 cells. (A) Flow cytometric analysis of cell cycle and apoptosis in cultured A20 cells. A20 cells without (A20 control) or with empty (vector control) or Lxn-containing vector (Lxn vector) were cultured for 21 days. BrdU and 7AAD staining and flow cytometric analysis was used for detecting different phases of the cell cycle (G0/G1, S, G2/M) and apoptotic (A) and necrotic (N) population. A representative flow cytometric file from Day 5 of culture is shown. (B): Down-regulation of anti-apoptotic genes by Lxn overexpression. Apoptosis PCR arrays were performed on A20 cells infected with empty or Lxn-expressing vectors. Left panel shows the fold changes in the mRNA expression of several selected genes (as indicated in X axis) in Lxn-overexpressing cells compared to the control. Western blot confirms the decreased expression of Bcl-2 and two isoforms of Pim-2 at the protein level in Lxn-overexpressing cells (arrowhead). The other apoptosis-related genes, such as Bcl-xl, Bax and Bad, did not show significant difference in mRNA and protein expression. (C) The growth curve of A20 cells treated with graded doses of potato carboxypeptidase inhibitor (PCPI). No significant difference is detected between the control and any of the doses of PCPI. (D) Internalization of PCPI to cytosol of A20 cells. A20 cells were cultured with fluorescein isothiocyanate (FITC) labeled PCPI. Shown is a three-color micrograph with PCPI in green (FITC), the B220 lymphoid cell surface marker in red (phycoerythrin, PE), the nucleus in blue (DAPI), and merged image of red and green as indicated. doi:10.1371/journal.pone.0044979.g004 the mononuclear white blood cell compartment. Resulting leukocytes from marrow or blood were then used for immunoaffinity selection and flow cytometric sorting. For CD34+ cell selection, the Miltenyi immunoaffinity device (VarioMACS) was used according to the manufacturer’s instructions (Miltenyi Biotech, Auburn, CA), and further purified by immune-staining with anti-CD34 antibodies (Phar- mingen, San Diego, CA) and sorting in a triple-laser FACSVantage flow cytometer (Becton Dickinson Immunocyto- metry Systems, San Jose, CA). In some cases, leukocytes were cryopreserved at a concentration of 56107 cells/mL in freezing medium consisting of Iscoves modified Dulbecco medium (IMDM), 40% fetal bovine serum (FBS), and 10% dimethyl sulfoxide (DMSO). Quantitative Real-time PCR To measure the expression of Lxn in leukemic cells, quantitative real-time PCR was performed. Identical numbers (200,000) of cells were used for total RNA extraction using RNeasy Mini kit (QIAGEN, Valencia, CA) according to the manufacture’s instruction. Isolated total RNA was reverse transcribed into cDNA using random hexamers in a TaqManH reverse transcrip- tion solution (PN N8080234) and stored at 220uC. In real-time PCR reactions, primer and probe mix for LXN (human and mouse) were purchased from Applied Biosystems (Foster city, CA, USA). TaqManH human glyceraldehyde-3-phosphate dehydroge- nase (GAPDH) was served as an endogenous control to normalize LXN expression. PCR reactions were set up according to manufacturer’s instructions using TaqManH universal PCR master mix (PN 4304437). Analyses of gene expression were performed in Figure 5. Decreased Lxn expression in stem/progenitor-enriched CD34+ cells in patients with leukemia and lymphoma. (A) Lxn mRNA expression in bone marrow and peripheral blood CD34+ cells from leukemia and lymphoma patients and normal individuals. Lxn mRNA level was measured by quantitative real-time PCR and shown as mean (61 SD) (n = 12). Normal samples were derived from cord blood (CB), bone marrow (BM) of young at 31 (Y1) and 39 (Y2) years old and old at 85 (O1) and 97 (O2) years old people. The patient samples include acute myeloid leukemia (AML), T cell pro-lympho leukemia (Pctl), plasma cell leukemia (PCL), acute T cell lymphoma (ATLL) and acute lymphoid leukemia (ALL, preB phenotype). (B) LXN protein expression in bone marrow and peripheral blood CD34+ cells from leukemia and lymphoma patients and normal individuals. Western blot was performed on the corresponding samples shown in panel (A) plus five more samples, including two young (Y3 and Y4; at 40 and 31 years old respectively), two old (O3 and O4; at 80 and 87 years old respectively) and chronic myeloid leukemia (CML). The blots (bottom) and their quantification (top) profiles demonstrate the significantly decreased Lxn protein level in leukemic CD34+ and lymphoma cells (P = 0.03). doi:10.1371/journal.pone.0044979.g005 PLOS ONE | www.plosone.org 9 September 2012 | Volume 7 | Issue 9 | e44979