Aberrant overexpression of an epithelial marker, 14-3-3σ, in a subset of hematological malignancies-4

<p><b>Copyright information:</b></p><p>Taken from "Aberrant overexpression of an epithelial marker, 14-3-3σ, in a subset of hematological malignancies"</p><p>http://www.biomedcentral.com/1471-2407/7/217</p><p>BMC Cancer 2007;7():217-217.</p><p>Published online 25 Nov 2007</p><p>PMCID:PMC2222637.</p><p></p>rkat cells (an open square). Dotted line denotes mean plus 2 SDs (2.17) of controls and a mean for each disease or cell line is shown as a horizontal bar. The vertical scales are different between the lower and higher levels. Open circles denote patients with p53 mutations identified by RT-PCR-SSCP analysis. Controls include 5 PBMNC, 3 BMMNC, 2 reactive lymph nodes; CML, 7 patients with blast crisis (BC) and 1 patient with chronic phase (CP) of chronic myeloid leukemia; AML, 12 patients with acute myeloid leukemia; ALL, 16 patients with acute lymphoid leukemia including 3 patients with Burkitt leukemia/lymphoma; ATL, 8 patients with adult T-cell leukemia/lymphoma (1 chronic, 1 acute, and 6 lymphoma variants); CLL, 5 patients with chronic lymphocytic leukemia/small lymphocytic lymphoma and 3 patients with prolymphocytic leukemia; DLBCL, 27 patients with diffuse large B-cell lymphoma; FL, 29 patients with follicular lymphoma; other NHL, 4 patients with T-cell lymphoma, 2 patients with mantle cell lymphoma and 3 patients with unclassified lymphoma; PCD, 12 patients with plasma cell dyscrasias including 1 patient with plasma cell leukemia, 4 patients with Waldenström's macroglobulinemia and 7 patients with multiple myeloma; cell lines include 14 myeloid, 15 T-cell, and 10 B-cell lines (all listed in reference [23]) and an epithelial cell line, HeLa (an open triangle). Asterisks denote statistical significance by Scheffe test after combining CML BC, AML and ALL as a group of acute leukemia and DLBCL, FL and other NHL as a group of NHL: * P < 0.05, ** P < 0.01

Aberrant overexpression of an epithelial marker, 14-3-3σ, in a subset of hematological malignancies-2

<p><b>Copyright information:</b></p><p>Taken from "Aberrant overexpression of an epithelial marker, 14-3-3σ, in a subset of hematological malignancies"</p><p>http://www.biomedcentral.com/1471-2407/7/217</p><p>BMC Cancer 2007;7():217-217.</p><p>Published online 25 Nov 2007</p><p>PMCID:PMC2222637.</p><p></p>ose gel. Representative results are shown. The 14-3-3σ mRNA expression levels determined by real-time RT-PCR are shown below. M and U denotes methylated and unmethylated alleles, respectively. PCL denotes plasma cell leukemia; MM, multiple myeloma; CML-CP, chronic phase of chronic myeloid leukemia; PLL, prolymphocytic leukemia

Aberrant overexpression of an epithelial marker, 14-3-3σ, in a subset of hematological malignancies-3

<p><b>Copyright information:</b></p><p>Taken from "Aberrant overexpression of an epithelial marker, 14-3-3σ, in a subset of hematological malignancies"</p><p>http://www.biomedcentral.com/1471-2407/7/217</p><p>BMC Cancer 2007;7():217-217.</p><p>Published online 25 Nov 2007</p><p>PMCID:PMC2222637.</p><p></p> of cell-cycle inhibitors, expression levels are plotted in scattered plots. Open circles denote patients with p53 mutations identified by RT-PCR-SSCP analysis. Correlation coefficients for p53 wild-type and mutated patients are: 14-3-3σ vs. ARF, -0.074 and -0.129; 14-3-3σ vs. CDKN2A, -0.033 and -0.391; ARF vs. CDKN2A, -0.044 and -0.177, respectively

Primers used for real time based PCR.

<p>Primers used for real time based PCR.</p

Primers used for RT-PCR.

<p>Primers used for RT-PCR.</p

Phenotypes of GABA_BR1-null mice.

<p>Plasma was collected from blood of male mice at 4 weeks old, followed by measurement of (A) leptin and (B) insulin using ELISA kits. Various tissues were dissected from male mice at 4 weeks old, followed by measurement of wet weights of (C) WAT and (D) other tissues for calculation of the percentage over body weight. (E) Mice were fed with powder diets for 1 week, followed by measurement of the accumulated amount of food intake during 1 week. Open-field tests were carried out using male mice at 4 weeks old for (F) the number of crossing and (G) the number of rearing. (H) Body temperature was measured at the rectum in mice. (I) Mice were fasted for 15 to 16 h and then injected ip with 2 g/kg glucose, followed by determination of blood glucose levels 15 to 120 min after the injection. (J) Basal blood glucose levels were measured in male mice at 4 weeks old. Values are the mean ± S.E. from different experiments shown in the figure. *P<0.05; **P<0.01, significantly different from each control value obtained in WT mice. B.W., body weight; Gb1, GABA_BR1.</p

Expression profiles of GABAergic signaling molecules.

<p>(A) Total RNA was extracted from 3T3-L1 cells cultured for 2 to 8 days and epididymal WAT of male mice, followed by RT-PCR using specific primers for each molecule. (B) Both 3T3-L1 cells and EF were cultured until confluence, followed by detection of GABA_BR1 subunit protein on immunoblotting analysis. Mouse whole brain was used as a positive control. Expression of mRNA and corresponding protein for GABA_BR1 subunit was constitutively seen in adipocytic cells.</p

Transfection with GABA_BR1 siRNA in 3T3-L1 cells.

<p>(A) 3T3-L1 cells transfected with si-control and si-GABA_BR1 were cultured for 3 days, followed by immunoblotting for GABA_BR1 subunit. (B) 3T3-L1 cells transfected with siRNA were cultured for 2 days in the presence of inducers of adipocytic differentiation, followed by real time-PCR for GABA_BR1 subunit, leptin and PGC1α. (C) 3T3-L1 cells were transfected with the luciferase vector containing leptin promoter together with siRNA, followed by culture in either the presence or absence of inducers such as insulin, DEX and IBMX for 2 days and subsequent determination of luciferase activity. (D) 3T3-L1 cells were transfected with the luciferase vector containing PGC1α promoter along with siRNA, followed by culture for 3 days and subsequent determination of luciferase activity. Values are the mean ± S.E. from 3 different experiments. *P<0.05; **P<0.01, significantly different from each control value. ^††P<0.01, significantly different from the value obtained in control cells cultured in the presence of inducers. Knockdown by siRNA of GABA_BR1 subunit led to decreased leptin and increased PGC1α mRNA expression levels through modulation of transactivation of corresponding genes. Gb1, GABA_BR1.</p

Lack of effects of GABA_BR ligands on adipocytic differentiation.

<p>(A) 3T3-L1 cells were transfected with the luciferase vector containing 4 tandem copies of CRE site, followed by exposure to the GABA_BR agonist baclofen, the GABA_BR antagonist CPG46381 or forskolin for 24 h and subsequent determination of luciferase activity. 3T3-L1 cells were cultured for 8 to 16 days, in either the presence or absence of baclofen and the antagonist saclofen, followed by Oil red O staining. Typical micrographic pictures are shown in the panel (B), while in the panel (C) quantitative data are shown as the mean ± S.E in 5 independent determinations. (D) EF was isolated from WT and GABA_BR1-null mice, followed by culture for 5 to 16 days and subsequent staining with Oil red O. Values are the mean ± S.E. from 6 to 11 different experiments. Neither agonist nor antagonist for GABA_BR exhibited significant effects on adipogenesis. Ba, baclofen; Gb1, GABA_BR1; Sa, saclofen.</p