Representative genes from microarray analysis.

*<p>Genes listed are those which showed more than 2.0-fold differences in Exp. 1 and 2 and more than 1.7-fold differences in Exp. 3 between <i>Tcl1</i>-expressing and -deficient ES cells.</p>**<p>Exp. 1: <i>Tcl1^−/−</i> #4 vs. <i>Tcl1^−/−</i>(CAG-<i>Tcl1</i>) #1.</p>***<p>Exp. 2: <i>Tcl1^−/−</i>(CAG-EGFP) #6 vs. <i>Tcl1^−/−</i>(CAG-<i>Tcl1</i>) #4.</p>****<p>Exp. 3: Mean values of relative gene expression in <i>Tcl1^−/−</i> #4 vs. wild-type and that in <i>Tcl1^−/−</i> #5 vs. wild-type.</p

Analysis of Akt and Wnt/β-catenin signaling in <i>Tcl1-</i>deficient and -overexpressing ES cells.

<p>(A) Western blot analysis of GSK, Akt, and β-catenin in wild-type (WT), <i>Tcl1^−/−</i> (KO) #2 and #4, <i>Tcl1^−/−</i>(CAG-<i>Tcl1</i>) #1 and #3, and <i>Tcl1^−/−</i>(CAG-EGFP) #5 ES cells. (B) Western blot analysis of active β-catenin, Oct3/4, and HSP90 in the cytoplasmic (C) and nuclear (N) fractions of wild-type (WT), <i>Tcl1^−/−</i> (KO) #2 and #4, and <i>Tcl1^−/−</i>(CAG-<i>Tcl1</i>) #10 and #1 ES cells. <i>Tcl1^−/−</i>(CAG-<i>Tcl1</i>) #10 and #1 were derived from <i>Tcl1^−/−</i> (KO) #2 and #4, respectively. Proper fractionation was confirmed by western blotting of Oct3/4 and HSP90, which localize to the nucleus and cytoplasm, respectively. Be8cause active β-catenin levels in the nuclear fractions were much lower than those in the cytoplasmic fractions, active β-catenin in the nuclear fractions was detected by approximately two-fold longer exposure compared with that in the cytoplasmic fractions. (C) TOPflash assay. <i>P</i> values of wild-type ES cells (WT) compared with <i>Tcl1^−/−</i>(CAG-<i>Tcl1</i>) #1 and #3 ES cells were less than 0.01. <i>P</i> values of <i>Tcl1^−/−</i> (KO) #4 and #5 ES cells compared with <i>Tcl1^−/−</i>(CAG-<i>Tcl1</i>) #1 and #3 ES cells were less than 0.02.</p

Effects of <i>Tcl1</i> deficiency and overexpression on ES-cell differentiation.

<p>The expression of representative stem and differentiation markers was examined by real time PCR in ES cells grown in LIF(+) culture (left half of each panel) and with EB formation (right half of each panel). For EB formation, trypsinized ES cells were seeded into a bacterial grade dish, and cultured for 12 days. Values are expressed as mean ± SEM of three technical replicates. ∗<i>P</i><0.05 and ∗∗<i>P</i><0.01 by Student’s t-test. <i>Tcl1^−/−</i> (KO) vs. wild-type (WT) or <i>Tcl1^−/−</i>(CAG-<i>Tcl1</i>) cells.</p

Targeted disruption of the murine <i>Tcl1</i> gene.

<p>(A) <i>Tcl1</i> gene structure and targeting vector. Arrows represent the forward and reverse primers (P1 and P2) used to screen the targeted ES clones, and arrowheads (H1 and H2) represent the primers used to identify homozygous knockout clones. (B) Identification of <i>Tcl1</i>+/+, +/−, and −/− ES cell clones by genomic PCR using H1 and H2. (C) Western blot analysis of Tcl1 and β-actin in wild-type (WT), <i>Tcl1^−/−</i> (KO) #2 and #4, <i>Tcl1^−/−</i>(CAG-<i>Tcl1</i>) #1 and #3, and <i>Tcl1^−/−</i>(CAG-EGFP) #5 ES cells. <i>Tcl1^−/−</i>(CAG-<i>Tcl1</i>) #1 and #3, and <i>Tcl1^−/−</i>(CAG-EGFP) #5 were derived from <i>Tcl1^−/−</i> (KO) #4.</p

Differentially expressed genes in <i>Tcl1</i><i>^−/−</i> ES cells compared with <i>Tcl1</i><i>^−/−</i>(CAG-<i>Tcl1</i>) and wild-type ES cells.

<p>(A) Real time PCR analysis of 2 genes that were shown to be upregulated by <i>Tcl1</i> expression in DNA microarray analysis. (B) Real time PCR analysis of 16 genes that were shown to be downregulated by <i>Tcl1</i> expression in DNA microarray analysis. Expression of these genes were compared among wild-type (WT), <i>Tcl1^−/−</i> (KO) #5, and <i>Tcl1^−/−</i>(CAG-<i>Tcl1</i>) #11 and #14 ES cells. <i>Tcl1^−/−</i>(CAG-<i>Tcl1</i>) #11 and #14 were derived from <i>Tcl1^−/−</i> (KO) #5. Values are expressed as means ± SEM of three technical replicates. Difference from <i>Tcl1^−/−</i> (KO) ES cells: ∗<i>P</i><0.05, ∗∗<i>P</i><0.01.</p

Effect of <i>Tcl1</i> expression on ES cell growth.

<p>(A) Cell proliferation assay. Cell proliferation between 24 and 48 hours of culture was analyzed by MTT assay. The proliferation rate of <i>Tcl1^−/−</i> (KO) was significantly lower than that of wild-type (WT) or <i>Tcl1^−/−</i>(CAG-<i>Tcl1</i>) cells. Values are expressed as means ± SD (left panel, n = 12 each; right panel, n = 8 each). Difference from <i>Tcl1^−/−</i> (KO) ES cells: ∗∗<i>P</i><0.01. (B) The percentage of cleaved caspase 3-positive cells was calculated for 9–13 areas selected at random (left panel, n = 9–13, 6312–14211 nuclei per cell line; right panel, n = 10, 3329–4538 nuclei per cell line). The percentage of cleaved caspase 3-positive apoptotic cells in <i>Tcl1^−/−</i> (KO) cells was significantly higher than that in wild-type (WT) or <i>Tcl1^−/−</i>(CAG-<i>Tcl1</i>) cells. Values are expressed as means ± SD. Difference from <i>Tcl1^−/−</i> (KO) ES cells: ∗<i>P</i><0.05, ∗∗<i>P</i><0.01. (C) Teratoma formation of wild-type (WT) (n = 9), <i>Tcl1^−/−</i> (KO) #4 (n = 6), <i>Tcl1^−/−</i>(CAG-EGFP) #5 (n = 3), and <i>Tcl1^−/−</i>(CAG-<i>Tcl1</i>) #1 (n = 3) and #3 (n = 5) ES cells. The weight of each teratoma is shown in grams. Horizontal bars indicate the mean of each sample. ∗<i>P</i><0.03. (D) Histological analysis of the teratomas derived from <i>Tcl1^−/−</i> (KO) #4 and #5 and <i>Tcl1^−/−</i>(CAG-<i>Tcl1</i>) #1 and #3 ES cells. No histological differences were recognizable between <i>Tcl1^−/−</i> (KO) and <i>Tcl1^−/−</i>(CAG-<i>Tcl1</i>) ES cells.</p