Journal: Nutrients

Article Title: Melatonin Efficacy in Obese Leptin-Deficient Mice Heart

doi: 10.3390/nu9121323

Figure Lengend Snippet: Pericardial adipose tissue evaluations. Photomicrographs showing pericardial adipocytes morphological feature through haematoxylin-eosin staining ( A – C ), anti-obesogenic adiponectin ( D – F ) and 4HNE ( G – I ) immunostainings of lean mice treated and not treated with melatonin ( A , D , G ), ob / ob mice ( B , E , H ) and ob / ob mice treated with melatonin ( C , F , I ). The graphs summarize adipocyte area ( L ), adiponectin ( M ) and 4HNE ( N ) immunopositivities. Lean mice treated or not with melatonin displayed similar “normal” pericardial adipose tissue features, so they are defined generically as “lean” mice. Note that ob / ob mice treated with melatonin showed a reduced white adipocytes area and inverse relationship between anti-diabetogenic adiponectin and 4HNE. Scale Bar = 20 µm. * p ≤ 0.05 significant vs. lean mice; ° p ≤ 0.05 significant vs. ob / ob mice.

Article Snippet: In the present study, we evaluated the following primary antibodies: monoclonal mouse antibodies against mitofusin2 (Mfn2) clone 6A8 (Abnova, Taipei, Taiwan) diluted 1:200; polyclonal rabbit antibodies against adiponectin (Abcam, Cambridge, UK) diluted 1:500; rabbit polyclonal antibodies against p62/SQSTM1 (MBL Ltd, Jasola, New Delhi, India) diluted 1:50 and rabbit polyclonal antibodies against 4 hydroxy-2-nonenal (4HNE) (Abcam, Cambridge, UK) diluted 1:400.

Techniques: Staining, Mouse Assay

Journal: Nutrients

Article Title: Melatonin Efficacy in Obese Leptin-Deficient Mice Heart

doi: 10.3390/nu9121323

Figure Lengend Snippet: Obesity alterations and melatonin protective effects at both heart and pericardial adipose tissue level. Leptin deficient ob / ob mice presented, in ventricular cardiomyocytes, mitochondria with abnormal large size, called mega-mitochondria, together with high level of both 4hydroxy-2-nonenal (4HNE), a marker of lipid peroxidation and of p62/SQSTM1, an index of aberrant autophagic flux but scarce degree of mitofusin2 (Mfn2), indicative of mitochondrial sufferance. Furthermore, the ob / ob mice showed, in pericardial adipose tissue, low level of adiponectin expression and higher level of 4HNE, respect lean mice. Interestingly, melatonin oral supplementation in the heart restored mitochondria shape and ultrastructure, enhanced Mfn2 expression and minimized 4HNE and p62/SQSTM1. In addition, at pericardial fat level, melatonin reduced adipocyte hypertrophy, increased adiponectin and reduced 4HNE expressions.

Article Snippet: In the present study, we evaluated the following primary antibodies: monoclonal mouse antibodies against mitofusin2 (Mfn2) clone 6A8 (Abnova, Taipei, Taiwan) diluted 1:200; polyclonal rabbit antibodies against adiponectin (Abcam, Cambridge, UK) diluted 1:500; rabbit polyclonal antibodies against p62/SQSTM1 (MBL Ltd, Jasola, New Delhi, India) diluted 1:50 and rabbit polyclonal antibodies against 4 hydroxy-2-nonenal (4HNE) (Abcam, Cambridge, UK) diluted 1:400.

Techniques: Marker, Expressing

Journal: PLoS ONE

Article Title: Additive Regulation of Adiponectin Expression by the Mediterranean Diet Olive Oil Components Oleic Acid and Hydroxytyrosol in Human Adipocytes

doi: 10.1371/journal.pone.0128218

Figure Lengend Snippet: Human SGBS adipocytes were pretreated with HT (1 h) (A), OA (48 h) or RSG (24 h) (B) at the concentrations indicated and then either treated with 10 ng/mL TNF-α (black-filled bars), or left untreated (open white bars), for 24 h. Adiponectin levels in the culture medium were determined by ELISA, and expressed as percent of unstimulated control (CTL). Bars represent means ± SD (n = 3). # p <0.05 versus CTL. * p <0.05 versus TNF-α. ** p <0.01 versus TNF-α.

Article Snippet: 3T3-L1 adipocytes grown in 24-well plates on coverslips (Thermanox, ProSciTech, Thuringowa, Queensland, Australia) were pretreated with HT or OA before TNF-α stimulation for 24 h. The specimens were fixed with cold acetone and then incubated overnight with a rabbit polyclonal antibody against adiponectin (Millipore, Billerica, MA, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Control

Journal: PLoS ONE

Article Title: Additive Regulation of Adiponectin Expression by the Mediterranean Diet Olive Oil Components Oleic Acid and Hydroxytyrosol in Human Adipocytes

doi: 10.1371/journal.pone.0128218

Figure Lengend Snippet: SGBS cells were pretreated with either HT, OA or cotreated with HT + OA before 10 ng/mL TNF-α stimulation for 24 h. (A) Adiponectin in the culture medium was determined by ELISA, and expressed as percent of unstimulated control (CTL). (B) Adiponectin intracellular protein levels were determined by Western analysis using antibodies against adiponectin. Western analysis under reducing and denaturing condition here reveals the 30 kDa adiponectin monomer. Adiponectin expression was normalized to β-actin, and expressed as percent of unstimulated control (CTL). Data are means ± SD (n = 3). # p <0.05 versus CTL. * p <0.05 versus TNF-α alone. † p <0.05 versus each compound + TNF-α.

Article Snippet: 3T3-L1 adipocytes grown in 24-well plates on coverslips (Thermanox, ProSciTech, Thuringowa, Queensland, Australia) were pretreated with HT or OA before TNF-α stimulation for 24 h. The specimens were fixed with cold acetone and then incubated overnight with a rabbit polyclonal antibody against adiponectin (Millipore, Billerica, MA, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Control, Western Blot, Expressing

Journal: PLoS ONE

Article Title: Additive Regulation of Adiponectin Expression by the Mediterranean Diet Olive Oil Components Oleic Acid and Hydroxytyrosol in Human Adipocytes

doi: 10.1371/journal.pone.0128218

Figure Lengend Snippet: SGBS cells were pretreated with either HT, OA or cotreated with HT + OA before 10 ng/mL TNF-α stimulation for 24 h. Adiponectin mRNA levels were determined by qPCR and normalized to 18S RNA. Data are expressed as fold induction over unstimulated control (CTL). Data are means ± SD (n = 3). # p <0.05 versus CTL. * p <0.05 versus TNF-α alone. † p <0.05 versus each compound + TNF-α.

Article Snippet: 3T3-L1 adipocytes grown in 24-well plates on coverslips (Thermanox, ProSciTech, Thuringowa, Queensland, Australia) were pretreated with HT or OA before TNF-α stimulation for 24 h. The specimens were fixed with cold acetone and then incubated overnight with a rabbit polyclonal antibody against adiponectin (Millipore, Billerica, MA, USA).

Techniques: Control

Journal: PLoS ONE

Article Title: Additive Regulation of Adiponectin Expression by the Mediterranean Diet Olive Oil Components Oleic Acid and Hydroxytyrosol in Human Adipocytes

doi: 10.1371/journal.pone.0128218

Figure Lengend Snippet: (A) SGBS cells were treated with 1 μmol/L HT, 10 μmol/L OA, or 1 μmol/L RSG in the absence or presence of the PPARγ antagonist GW9662 at 10 μmol/L (GW), and then stimulated with 10 ng/mL TNF-α for 24 h. Adiponectin levels in the culture medium were determined by ELISA, and expressed as percent of unstimulated control (CTL). Data are means ± SD (n = 3). # p <0.05 versus CTL. * p <0.05 versus TNF-α alone. † p <0.05 versus the compound-treated group without GW9662. (B) and (C) SGBS cells were treated with 1 μmol/L HT or 10 μmol/L OA before 10 ng/mL TNF-α stimulation for 24 h. (B) Whole-cell lysates were assayed by Western blotting using antibodies against PPARγ1, PPARγ2, and against β-actin, this last used as a loading control. Total PPARγ1 and PPARγ2 band intensities were normalized to β-actin, and are expressed as percent of unstimulated control (CTL). (C) Nuclear proteins were analyzed for PPARγ DNA-binding activity by ELISA as described in Methods. Data are expressed as percent of unstimulated control (CTL). (D) SGBS cells were treated with 1–10 μmol/L HT, or 10 μmol/L OA, or co-treated with OA + HT before 10 ng/mL TNF-α stimulation for 24 h. PPARγ mRNA levels were determined by qPCR and normalized to 18S RNA. Data are expressed as fold induction over unstimulated control (CTL). Bars represent means ± SD (n = 3). # p <0.05 versus CTL. * p <0.05 versus TNF-α. † p <0.05 versus each compound + TNF-α.

Article Snippet: 3T3-L1 adipocytes grown in 24-well plates on coverslips (Thermanox, ProSciTech, Thuringowa, Queensland, Australia) were pretreated with HT or OA before TNF-α stimulation for 24 h. The specimens were fixed with cold acetone and then incubated overnight with a rabbit polyclonal antibody against adiponectin (Millipore, Billerica, MA, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Control, Western Blot, Binding Assay, Activity Assay

Journal: PLoS ONE

Article Title: Additive Regulation of Adiponectin Expression by the Mediterranean Diet Olive Oil Components Oleic Acid and Hydroxytyrosol in Human Adipocytes

doi: 10.1371/journal.pone.0128218

Figure Lengend Snippet: SGBS cells were pretreated for 1 h with 10 μmol/L of the JNK inhibitor SP600125 (SP), the ERK1/2 inhibitor PD98059 (PD), or the p38 inhibitor SB203580 (SB), and then stimulated with 10 ng/mL TNF-α for 24 h. Culture media were analyzed for adiponectin by ELISA (A), and whole-cell lysates were assayed by Western blotting using antibodies against adiponectin (B) or PPARγ (C). Adiponectin and PPARγ expression were normalized to β-actin, and expressed as percent of unstimulated control (CTL). Bars represent means ± SD (n = 3). # p <0.05 versus CTL. * p <0.05 versus TNF-α.

Article Snippet: 3T3-L1 adipocytes grown in 24-well plates on coverslips (Thermanox, ProSciTech, Thuringowa, Queensland, Australia) were pretreated with HT or OA before TNF-α stimulation for 24 h. The specimens were fixed with cold acetone and then incubated overnight with a rabbit polyclonal antibody against adiponectin (Millipore, Billerica, MA, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Control

Journal: PLoS ONE

Article Title: Additive Regulation of Adiponectin Expression by the Mediterranean Diet Olive Oil Components Oleic Acid and Hydroxytyrosol in Human Adipocytes

doi: 10.1371/journal.pone.0128218

Figure Lengend Snippet: SGBS cells were treated with scrambled negative control siRNA (siControl), JNK1 siRNA (siJNK1), JNK2 siRNA (siJNK2), or JNK1 plus JNK2 siRNA, for 72 h. The mRNA expression levels of JNK1 and JNK2 were measured by qPCR, normalized to 18S RNA, and expressed as fold induction over scrambled negative control siRNA (A). JNK1 and JNK2 intracellular protein levels were assayed by Western blotting, normalized to β-actin, and expressed as percent of scrambled negative control siRNA (B). Bars represent means ± SD. # p <0.05 versus siControl. After 72 h of transfection, cells were stimulated with 10 ng/mL TNF-α for further 24 h. Adiponectin mRNA were determined by qPCR (C), while adiponectin intracellular and secreted protein levels were determined by Western analysis (D) and ELISA (E), respectively. Bars represent means ± SD. # p <0.05 versus siControl without TNF-α. * p <0.05 versus siControl with TNF-α.

Article Snippet: 3T3-L1 adipocytes grown in 24-well plates on coverslips (Thermanox, ProSciTech, Thuringowa, Queensland, Australia) were pretreated with HT or OA before TNF-α stimulation for 24 h. The specimens were fixed with cold acetone and then incubated overnight with a rabbit polyclonal antibody against adiponectin (Millipore, Billerica, MA, USA).

Techniques: Negative Control, Expressing, Western Blot, Transfection, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Additive Regulation of Adiponectin Expression by the Mediterranean Diet Olive Oil Components Oleic Acid and Hydroxytyrosol in Human Adipocytes

doi: 10.1371/journal.pone.0128218

Figure Lengend Snippet: Pre-treatment with HT and OA before TNF-α stimulation prevents JNK activation and restores PPARγ expression and activity and, as a consequence, adiponectin levels. A coherent interpretation of the findings is as follows: upon binding to the cognate receptor, TNF-α induces reactive oxygen species (ROS) production and triggers an inflammatory signaling cascade involving, among others, the activation of JNK, which mediates the degradation of PPARγ (a transcription factor implicated in adiponectin gene expression through a PPAR-responsive element (PPRE) in its promoter). As a result, adiponectin expression is downregulated. Arrow indicates stimulation. Line indicates inhibition. MKK: MAP kinase kinase; pJNK: phosphorylated JNK.

Article Snippet: 3T3-L1 adipocytes grown in 24-well plates on coverslips (Thermanox, ProSciTech, Thuringowa, Queensland, Australia) were pretreated with HT or OA before TNF-α stimulation for 24 h. The specimens were fixed with cold acetone and then incubated overnight with a rabbit polyclonal antibody against adiponectin (Millipore, Billerica, MA, USA).

Techniques: Activation Assay, Expressing, Activity Assay, Binding Assay, Gene Expression, Inhibition

Journal: Molecular Biology of the Cell

Article Title: Trafficking of the Menkes copper transporter ATP7A is regulated by clathrin-, AP-2–, AP-1–, and Rab22-dependent steps

doi: 10.1091/mbc.e12-08-0625

Figure Lengend Snippet: FIGURE 8: Expression of the Rab22aQ64L mutant inhibits ATP7A trafficking. HeLa cells transiently transfected with myc-tagged, constitutively active Rab22aQ64L were treated with CuCl2, fixed, and colabeled for ATP7A (green) and anti-myc to detect the Rab22a mutant (a), EEA1 (b), or golgin-97 (c) (all red). Cells expressing Rab22aQ64L (d) were treated with copper, followed by washout, and colabeled for ATP7A (green) and myc tag (red). Nuclei are labeled with DAPI (blue). Scale bar, 10 μm.

Article Snippet: Polyclonal antibodies against EEA1 (C45B10) and clathrin heavy chain (P1663) were from Cell Signaling Technology; GM130 has been described (Nelson et al., 1998); ATP7A (Steveson et al., 2003) was a kind gift from Betty Eipper (University of Connecticut Health Center, Farmington, CT).

Techniques: Expressing, Mutagenesis, Transfection, Labeling