11 research outputs found

    Physiological measurements of sham and OVX of WT and MCP-1-KO mice 12 weeks after operation.

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    <p>Data are expressed as mean ± SEM. ND, non-detectible. Differences between groups were analyzed by two-way ANOVA, followed by Bonferroni post-tests (Increased body weight, subcutaneous fat, visceral fat, serum H<sub>2</sub>O<sub>2</sub>, and blood insulin; <i>P</i><0.001, serum M-CSF; <i>P</i><0.01, blood glucose; <i>P</i><0.05, effect of surgery. Increased body weight, subcutaneous fat, and visceral fat; <i>P</i><0.001, serum M-CSF and blood insulin; <i>P</i><0.01, serum ROS; <i>P</i><0.05, effect of MCP-1). WT OVX vs. MCP-1-KO OVX;</p>*<p><i>P</i><0.05,</p>**<p><i>P</i><0.01,</p>***<p><i>P</i><0.001.</p

    MCP-1-deficiency decreased OVX-induced immune cell infiltration in AT.

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    <p>SVCs from visceral fat were extracted from WT (open bar) and MCP-1-KO mice (oblique-lined bar) 12 weeks after sham or OVX surgery. SVCs were labeled with conjugated Abs to CD11bF4/80 (A), CD11cF4/80 (B), CD4 (C), and CD8 (D) and quantified by flow cytometry. Data are expressed as mean ± SEM. Differences between groups were analyzed by two-way ANOVA, followed by Bonferroni post-tests (CD11bF4/80, CD11cF4/80, CD4; <i>P</i><0.01, CD8; <i>P</i><0.05, effect of surgery. CD11cF4/80; <i>P</i><0.05, effect of MCP-1). *, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001 compared with WT OVX mice. Similar results were obtained in three independent experiments.</p

    MCP-1-deficiency decreased CD11c-expressing cells via impairing the production of ROS and decreased activation of PLCγ2, Akt, and ERK upon M-CSF stimulation in BMM.

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    <p>BMMs from WT (open bar) and MCP-1-KO mice (oblique-lined bar) were incubated in the presence of M-CSF (30 ng/ml) with U73122 (10 µM), Akt inhibitor IV (0.3 µM), PD098059 (5 µM), DPI (50 nM), NAC (3 mM), or H<sub>2</sub>O<sub>2</sub> (300 µM) for 4 d (A). *, <i>P</i><0.05; ***, <i>P</i><0.001 compared with vehicle-treated WT cells. There was no significant difference between WT and MCP-1-KO cells except upon vehicle treatment. Treatment with U73122, Akt inhibitor IV, PD98059, DPI, NAC, or H<sub>2</sub>O<sub>2</sub> abolished the decrease in CD11cF4/80 observed in MCP-1-KO cells. BMMs were serum-starved for 8 h and stimulated with M-CSF for 1, 2, or 3 d (B). Phosphorylation of PLCγ2 was determined by Western blotting. Total protein level served as the loading control. Relative ratios of phosphorylated forms to total forms were plotted. **, <i>P</i><0.01; ***, <i>P</i><0.001 compared with WT cells. Intracellular levels of ROS upon stimulation in the presence of M-CSF (M) or/and MCP-1 with control IgG (3 µg/ml) or anti-MCP-1 Ab (3 µg/ml) for 2 d were determined in WT cells and MCP-1-KO cells using H2DCFDA (C, D). ROS levels were quantified by flow cytometry. *, <i>P</i><0.05; **, <i>P</i><0.01 compared with M-stimulated WT cells. No significant difference between WT and MCP-1-KO cells stimulated with M+MCP-1 (C). *, <i>P</i><0.05; ***, <i>P</i><0.001 compared with IgG-treated WT cells. No significant difference between IgG- and anti-MCP-1 Ab-treated MCP-1-KO cells (D). BMMs were transfected with sip47<sup>phox</sup> or scRNA. Downregulation of p47<sup>phox</sup> by siRNA was confirmed by RT-PCR and qPCR (E). The expression level obtained from scRNA-treated cells was set to be 1. After 24 h of transfection with siRNA, cells were stimulated with M-CSF for 2 d (mRNA) or 4 d (FACS) in order to determine CD11c (F) and for 2 d to measure ROS (G). *, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001 compared with scRNA-transfected WT cells. No significant difference was found in MCP-1-KO cells (F, G). Similar results were obtained in three independent experiments.</p

    The absence of MCP-1 reduced fat mass and improved metabolic perturbation induced by OVX.

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    <p>WT mice (open bar) and MCP-1-KO (oblique-lined bar) mice were subjected to OVX or sham surgery, and then held for 12 weeks. Whole body weight change up to 12 weeks after surgery (A) and average daily food intake (B) were measured. *, <i>P</i><0.05; WT OVX vs. MCP-1-KO OVX mice. Adipocyte volume was calculated from photograph of hematoxylin-eosin staining of visceral fat, assuming that an adipocyte is a sphere (magnification, ×200). Scale bar, 100 µm (C). Glucose clearance (D) and insulin sensitivity (E) were determined 12 weeks after sham or OVX, following an intraperitoneal injection of glucose (1 mg/kg) and insulin (0.75 munits/kg), respectively. AUC was measured for each group for D and E. *, <i>P</i><0.05, ***, <i>P</i><0.001; WT OVX vs. MCP-1-KO OVX. Data are expressed as mean ± SEM. Differences between groups were analyzed by two-way ANOVA, followed by Bonferroni post-tests (C, D) (adipocyte volume; <i>P</i><0.001, AUC for glucose tolerance; <i>P</i><0.01, effect of surgery. adipocyte volume; <i>P</i><0.001, AUC for glucose tolerance; <i>P</i><0.05, effect of MCP-1). *, <i>P</i><0.05; ***, <i>P</i><0.001 compared with WT OVX mice. Similar results were obtained in three independent experiments.</p

    β-lap in combination with IR induces apoptotic cell death via nuclear translocation of AIF.

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    <p>(A) NQO1<sup>+</sup>-MDA-MB-231 cells were treated with IR alone, β-lap alone or combination of IR and β-lap for the indicated times. The data represent a typical experiment conducted three times with similar results. (B) NQO1<sup>+</sup>-MDA-MB-231 cells were treated with IR alone, β-lap alone or combination of IR and β-lap for 12 h. Cytosolic fractions from NQO1<sup>+</sup>-MDA-MB-231 cells were prepared and subjected to Western blot analysis. The data are representative a typical experiment conducted three times. The data represent a typical experiment conducted three times with similar results. (C) NQO1<sup>+</sup>-MDA-MB-231 cells were treated with combination of IR and β-lap for 12 h in the presence or absence of z-VAD-fmk. The data represent a typical experiment conducted three times with similar results. (D) NQO1<sup>+</sup>-MDA-MB-231 cells were treated with IR alone, β-lap alone or combination of IR and β-lap for 24 h in the presence or absence of z-VAD-fmk (30 µM). After 24 h, the percentage of the cells with sub-G1 DNA content was determined by flow cytometry. Results from three independent experiments are expressed as means ± SEMs. (E) NQO1<sup>+</sup>-MDA-MB-231 cells were treated with IR alone, β-lap alone or combination of IR and β-lap for 24 h. Nuclear fractions from NQO1<sup>+</sup>-MDA-MB-231 cells were prepared and subjected to Western blot analysis. The data are representative a typical experiment conducted three times. (F) NQO1<sup>+</sup>-MDA-MB-231 cells were treated with IR alone, β-lap alone or combination of IR and β-lap for 24 h in the presence or absence of siRNA targeting AIF. After 24 h, the percentage of the cells with sub-G1 DNA content was determined by flow cytometry. Results from three independent experiments are expressed as means ± SEMs. (G) NQO1<sup>+</sup>-MDA-MB-231 cells were treated with combination of IR and β-lap for 24 h in the presence or absence of NAC, PD98059, Sal or SP600125. Nuclear fractions were prepared and subjected to Western blot analysis. The data are representative a typical experiment conducted three times.</p

    β-lap in combination with IR induces positive feedback regulation between ERK and ROS.

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    <p>(A) NQO1<sup>+</sup>-MDA-MB-231 cells were treated with IR alone, β-lap alone or combination of IR and β-lap for 30 min in the presence or absence of NAC. The data represent a typical experiment conducted three times with similar results. (B) NQO1<sup>+</sup>-MDA-MB-231 cells were treated with IR alone, β-lap alone or combination of IR and β-lap for 3 h in the presence or absence of siRNA targeting ERK1/2 or JNK2. After 3 h, the cells were incubated with 10 µM H2DCF-DA for 30 min and then analyzed by flow cytometry. Results from three independent experiments are expressed as means ± SEMs. (C) NQO1<sup>+</sup>-MDA-MB-231 cells were treated with IR alone, β-lap alone or combination of IR and β-lap for 30 min in the presence or absence of PD98059 or SP600125. The data represent a typical experiment conducted three times with similar results.</p

    The induction of ER stress by combined treatment with IR and β-lap is required for JNK activation.

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    <p>(A) NQO1<sup>+</sup>-MDA-MB-231 cells were treated with IR alone, β-lap alone or combination of IR and β-lap for the indicated times. Cell lysates were subjected to Western blot analysis. The data represent a typical experiment conducted three times with similar results. (B) NQO1<sup>+</sup>-MDA-MB-231 cells were treated with IR alone, β-lap alone or combination of IR and β-lap for 24 h in the presence or absence of Sal (10 µM). After 24 h, the percentage of cells with sub-G1 DNA content was determined by flow cytometry. Results from three independent experiments are expressed as means ± SEMs. (C) NQO1<sup>+</sup>- MDA-MB-231 cells were treated with IR alone β-lap alone or combination of IR and β-lap for 3 h in the presence or absence of Sal (10 µM). After 3 h, the cells were incubated with 10 µM H2DCF-DA for 30 min and then analyzed by flow cytometry. Results from three independent experiments are expressed as means ± SEMs. (D) NQO1<sup>+</sup>-MDA-MB-231 cells were treated with IR alone, β-lap alone or combination of IR and β-lap for 30 min in the presence or absence of NAC. The data represent a typical experiment conducted three times with similar results. (E) and (F) NQO1<sup>+</sup>-MDA-MB-231 cells were treated with IR alone, β-lap alone or combination of IR and β-lap for 30 min in the presence or absence of Sal, SP600125 or PD98059. The data represent a typical experiment conducted three times with similar results.</p

    β-lap in combination with IR rapidly activates ERK and JNK.

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    <p>(A) NQO1<sup>−</sup> or NQO1<sup>+</sup>-MDA-MB-231 cells were treated with IR alone, β-lap alone or combination of IR and β-lap for the indicated times. The data represent a typical experiment conducted three times with similar results. (B) and (C) NQO1<sup>+</sup>-MDA-MB-231 cells were treated with IR alone, β-lap alone or combination of IR and β-lap for 24 h in the presence or absence of PD98059 (30 µM), SP600125 (30 µM), SB203580 (30 µM), or siRNA targeting ERK1/2 or JNK2. The percentage of cells with sub-G1 DNA content was determined by flow cytometry. Results from three independent experiments are expressed as means ± SEMs.</p

    β-lap induces radiosensitization in an NQO1-dependent manner.

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    <p>(A) NQO1<sup>−</sup> or NQO1<sup>+</sup>-MDA-MB-231 cells were treated with various concentrations of β-lap. Cells were allowed to grow for 10 to 14 days and were stained with 0.5% crystal violet and scored for colony formation. Results from three independent experiments are expressed as means ± SEM. *Significant difference between NQO1<sup>−</sup>- and NQO1<sup>+</sup>-MDA-MB-231 cells after β-lap treatment at p<0.05. (B) Cells were treated with 2 µM β-lap and then exposed to increasing doses of IR 30 min after treatment with β-lap. After 14 days, cells were scored for colony formation. Results from three independent experiments are expressed as means ± SEMs. (C) Cells were treated with IR alone, β-lap alone or the combination of IR and β-lap for the indicated times. The percentage of cells with sub-G1 DNA content was determined by flow cytometry. Results from three independent experiments are expressed as means ± SEMs.</p

    β-lap in combination with IR enhances ROS generation and leads to apoptotic cell death.

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    <p>(A) and (B) Cells were treated with IR alone, β-lap alone or combination of IR and β-lap for the indicated times. After 3 h, the cells were incubated with 10 µM H2DCF-DA and 4 µM DHE, respectively, for 30 min and then analyzed by flow cytometry. Results from three independent experiments are expressed as means ± SEMs. (C) NQO1<sup>+</sup>-MDA-MB-231 cells were treated with IR alone, β-lap alone or combination of IR and β-lap for 24 h in the presence or absence of NAC (10 mM). After 24 h, the percentage of cells with sub-G1 DNA content was determined by flow cytometry. Results from three independent experiments are expressed as means ± SEMs. (D) NQO1<sup>+</sup>-MDA-MB-231 cells were treated with combination of IR (2 Gy) and β-lap (2 µM) in the presence or absence of NAC (10 mM). Cells were allowed to grow for 10 to 14 days and were stained with 0.5% crystal violet and scored for colony formation. Results from three independent experiments are expressed as means ± SEM. *Significant difference between cells in the presence or absence of NAC after combined treatment with IR and β-lap, at p<0.05.</p
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