Effect of AMPK inhibitor on CE-stimulated glucose uptake and ACC, LKB1 phosphorylation in 3T3-L1 adipocytes.

<p>3T3-L1 adipocytes were serum-starved for 16 h. For the inhibitor treatment, cells were pretreated with 200 µM Compound C (Com.C), an inhibitor of AMPK, for 20 min, after which the medium was changed to fresh medium containing 100 nM insulin or 30 µg/ml CE. The cells were then incubated for another 30 min. A: Cell lysates were analyzed by Western blotting using anti-phospho-AMPK and anti-AMPK antibodies. The data represent the mean ± S.E. of 5 different experiment *: <i>p</i><0.05 <i>vs</i> control; ‡: <i>p</i><0.05 <i>vs</i> CE; ns: indicates no significant difference (Bonferroni test). B: Glucose uptake was measured as described in the Materials and Methods section. Each value represents the mean ± S.E. of 3 different experiments. *: <i>p</i><0.05 vs control; †: <i>p</i><0.05 <i>vs</i> CE; ns: indicates no significant difference (Bonferroni test). C, D: Cell lysates were analyzed by Western blotting using anti-phospho-ACC, anti-phospho- LKB1, anti-ACC, and anti-LKB1 antibodies. The data represent the mean ± S.E. of 5 different experiments. *: <i>p</i><0.05 <i>vs</i> control; ns: indicates no significant difference (Bonferroni test).</p

Effect of inhibitors of insulin signaling kinases on CE-mediated glucose uptake and phosphorylation of AMPK.

<p>3T3-L1 adipocytes were serum-starved for 16 h. For the inhibitor treatment, the cells were pretreated with 60 µM AG1024 for 1 h. The medium was then changed to fresh medium containing 100 nM insulin or 30 µg/ml CE and thereafter stimulated for another 30 min. A: Glucose uptake was measured as described in Materials and Methods section. Each value represents the mean ± S.E. of 3 different experiments. B: Cell lysates were analyzed by Western blotting using anti-phospho-Akt and anti-Akt antibodies. C: Cell lysates were analyzed by Western blotting using anti-phospho-AMPK anti-AMPK antibodies. The data represent the mean ± S.E. of 5 different experiments. *, <i>p</i><0.05 <i>vs</i> control; †, <i>p</i><0.05 <i>vs</i> insulin; ‡, <i>p</i><0.05 <i>vs</i> CE; §, <i>p</i><0.05 <i>vs</i> insulin+CE; ns: indicates no significant difference (Bonferroni test).</p

Effect of LKB1 knockdown on AMPK phosphorylation and CE-stimulated glucose uptake.

<p>Differentiated C2C12 cells (A) were transfected with LKB1 siRNA for 24 h. Two different fields of observation are shown. B: Quantitative real-time PCR was used to determine the mRNA expression levels of LKB1 by the C2C12 cells that had been treated with LKB1 siRNA. The data represent the mean ± S.E. of 3 different experiments. **: <i>p</i><0.01 (Student’s t test). C: Effect of siRNA duplexes designed to reduce LKB1 expression on the LKB1 protein levels and on the phosphorylation of LKB1 in C2C12 myotube cells examined by Western blotting. These silencing effects were observed even in the culture at 24 h after the transfection. The data represent the mean ± S.E. of 4 different experiments. *: <i>p</i><0.05 <i>vs</i> control; †: <i>p</i><0.05 <i>vs</i> CE; ns: indicates no significant difference (Bonferroni test). D: CE (30 µg/ml) was added or not to LKB1 siRNA-transfected cells or negative control siRNA-transfected cells, which were then incubated for 30 min and thereafter harvested and analyzed by Western blotting using anti-phospho-AMPK, and anti-AMPK antibodies. The data represent the mean ± S.E. of 3 different experiments. *: <i>p</i><0.05 <i>vs</i> control; †: <i>p</i><0.05 <i>vs</i> CE; ns: indicates no significant difference (Bonferroni test). E: Differentiated C2C12 cells were transfected or not with LKB1 siRNA or negative control siRNA for 24 h and then treated or not with CE (30 µg/ml) for 30 min. The glucose uptake was measured by the method as described in the Materials and Methods section. The data represent the mean ± S.E. of 3 different experiments. *: <i>p</i><0.05 <i>vs</i> control; †: <i>p</i><0.05 <i>vs</i> CE; ns: indicates no significant difference (Bonferroni test).</p

Effect of CE administration on body weight and plasma insulin concentrations.

<p>Values are mean ± S.E. of 7 different rats.</p

Effect of CE administration on glucose and insulin intolerance in type 2 diabetic rats.

<p>A: For oral glucose tolerance test (OGTT), the rats were fasted for 6 h prior to performing the test. Glucose (1.2 g/kg b.w.) was orally administered and the blood-glucose concentration was measured by using the blood obtained from a tail vein at the indicated time points after the oral glucose loading. B: The area under the curve (AUC) of the OGTT for A. C: For insulin tolerance test (ITT), rats were fasted for 4 h. Then insulin (0.75 U/kg b.w.) was intraperitoneally injected and the blood-glucose concentration was measured as described above. D: The area under the curve (AUC) of the ITT for C. Data were analyzed by Bonferroni test. (n = 5–10 for each group, mean ± S.E.). *: <i>p</i><0.05 <i>vs</i> LETO; †: <i>p</i><0.05 <i>vs</i> OLETF; ns: indicates no significant difference.</p

Cell cycle progression of HT-29 cells cultured in the presence of alk(en)yl trisulfides having various structures

HT-29 cells were cultured in the presence or absence of alkyl trisulfides (dimethyl trisulfide, diethyl trisulfide, DPTS, dibutyl trisulfide and dipentyl trisulfide), alkenyl trisulfides (DATS, dibutenyl trisulfide and dipentenyl trisulfide) or mixed ank(en)yl trisulfide (allyl methyl trisulfide) at a concentration of 10 μM for 12 h. Then, the cell cycle distribution in G/M phase was analyzed by using a flow cytometer, as described in Materials and Methods (panel ). Values are the mean ± SE of three independent experiments. ** ( <p><b>Copyright information:</b></p><p>Taken from "Alkenyl group is responsible for the disruption of microtubule network formation in human colon cancer cell line HT-29 cells"</p><p></p><p>Carcinogenesis 2008;29(7):1400-1406.</p><p>Published online 29 May 2008</p><p>PMCID:PMC2500214.</p><p></p

Induction of mitotic arrest by DATS through the inhibition of mitotic spindle formation

Detection of Ser10-phosphorylated histone H3, a sensitive marker for cells at the M phase, was detected in HT-29 cells cultured with DATS (10 μM) for 12 h by using a flow cytometer (panel ). Spindle formation of HT-29 cells cultured with vehicle or DATS (10 μM) was assessed by the immunofluorescence method using anti-β-tubulin antibody, as described in Materials and Methods (green; panel ). The nucleus was counterstained with propidium iodide (magenta). Scale bar 20 μm.<p><b>Copyright information:</b></p><p>Taken from "Alkenyl group is responsible for the disruption of microtubule network formation in human colon cancer cell line HT-29 cells"</p><p></p><p>Carcinogenesis 2008;29(7):1400-1406.</p><p>Published online 29 May 2008</p><p>PMCID:PMC2500214.</p><p></p

BSO enhanced and sustained the DATS-induced cell cycle arrest but had little influence on DPTS-treated cells

HT-29 cells were pretreated with 500 μM BSO for 24 h and then the cells were treated with 10 μM DATS ( and ) or DPTS ( and ) for the times as indicated. The cell cycle distribution of the cells was analyzed by using a flow cytometer.<p><b>Copyright information:</b></p><p>Taken from "Alkenyl group is responsible for the disruption of microtubule network formation in human colon cancer cell line HT-29 cells"</p><p></p><p>Carcinogenesis 2008;29(7):1400-1406.</p><p>Published online 29 May 2008</p><p>PMCID:PMC2500214.</p><p></p