Journal: PLoS Genetics

Article Title: Congenital lipodystrophy induces severe osteosclerosis

doi: 10.1371/journal.pgen.1008244

Figure Lengend Snippet: A) Transplanted fat depots 3 months after surgery. B) Fat depot weight 3 months after transplantation. C) Serum leptin and adiponectin of FF mice 3 months after fat depot transplantation. WT and non-transplanted FF mice serve as control. D) μCT analysis of trabecular bone volume and bone mineral density of distal femurs of FF mice 3 months after sham operation or transplantation of fat derived from WT or adipokine-deficient mice. Data are presented as mean ± SD. **p<0.01; *** p<0.001 as determined by ANOVA with Holm-Sidak's post hoc analysis for multiple comparisons test. D) comparison with FF Sham except where detailed.

Article Snippet: The primary antibody cocktail contained rat anti-mouse CD45-BUV395 (BD Horizon, clone 30-F11, final dilution factor 1:200), rat anti-mouse TER-119-APC (BioLegend, clone TER-119, 1:200), rat anti-mouse CD41-BV421 (BioLegend, clone MWReg30, 1:300), rat anti-mouse/human CD11b (BioLegend, clone M1/70, 1:400), and rat-anti mouse Leptin receptor (LepR)-biotin (R&D Systems, polyclonal, 1:50) in Brilliant Stain Buffer (BD Biosciences) containing 10 μg/mL FcBlock.

Techniques: Transplantation Assay, Control, Derivative Assay, Comparison

Journal: Kidney international

Article Title: Adipose tissue explants and MDCK cells reciprocally regulate their morphogenesis in coculture.

doi: 10.1038/ki.2010.68

Figure Lengend Snippet: Figure 8 | Adipokine treatment. Effects of leptin (b–e), its antibody (insets in b–e), and combination of leptin and adiponection (f) on morphology of Madin–Darby canine kidney (MDCK) cells at 7 days in culture. (a) (Control), MDCK cells without leptin and adiponectin treatment show a flat shape with cytoplasm and flattened nuclei. (b and c) 20 (b) or 50 ng/ml (c) leptin does not affect the morphology of MDCK cells. (d and e) 100 (d) or 1000 ng/ml (e) leptin allows MDCK cells to develop a tall and large columnar shape, with clear cytoplasm and vertically and basally situated nuclei. The leptin-induced morphology is similar to the adipose tissue fragment (ATF)- induced morphology of MDCK cells. Leptin antibody (L-ab, 25 mg/ml) abolishes the MDCK cell morphology induced by 100 (inset in d) or 1000 ng/ml leptin (inset in e), although the antibody does not affect the morphology of cells at the concentration of 20 (inset in b) or 50 ng/ml (inset in c) leptin. (f) Adiponectin (10 mg/ml) abolishes the MDCK cell morphology induced by 1000 ng/ml leptin.

Article Snippet: To examine the specificity of a leptin-induced effect on the morphology of MDCK cells, 25 mg/ml of rabbit polyclonal leptin antibody against rat leptin (BioVendor LLC, Candler, NC, USA) was added to the 20, 50, 100, or 1000 ng/ml leptin-treated cultures of MDCK cells alone.

Techniques: Control, Concentration Assay

Journal: Kidney international

Article Title: Adipose tissue explants and MDCK cells reciprocally regulate their morphogenesis in coculture.

doi: 10.1038/ki.2010.68

Figure Lengend Snippet: Figure 9 | Adiponectin and leptin production of adipose tissue fragments (ATFs) in the absence or presence of Madin–Darby canine kidney (MDCK) cells in the supernatants of outer and inner dishes at 7 days in culture. In the cultures of ATFs alone, adiponectin and leptin are detected. Their concentration is not significantly different between the outer and inner dishes (P40.05). In the supernatants of outer dishes, adiponectin concentrations in the cocultures of ATFs and MDCK cells are significantly higher than those in the cultures of ATFs alone (Po0.05), whereas leptin concentrations are not significantly different between cultures of ATFs with and without MDCK cells (P40.05). In cocultures of ATFs and MDCK cells, adiponectin (Po0.01) and leptin (Po0.05) concentrations in the inner dishes were significantly lower than those in the outer dishes.

Article Snippet: To examine the specificity of a leptin-induced effect on the morphology of MDCK cells, 25 mg/ml of rabbit polyclonal leptin antibody against rat leptin (BioVendor LLC, Candler, NC, USA) was added to the 20, 50, 100, or 1000 ng/ml leptin-treated cultures of MDCK cells alone.

Techniques: Concentration Assay

Journal: FEBS letters

Article Title: Similarities and differences in the transcriptional regulation of the leptin gene promoter in gastric and adipose cells.

doi: 10.1016/j.febslet.2005.02.031

Figure Lengend Snippet: Fig. 2. Hormonal regulation of leptin secretion in gastric MKN-74 and in adipose 3T3-L1 cells. Gastric MKN-74 (A) and differentiated adipose 3T3-L1 cells (B) were treated for 24 h with or without 10 nM CCK8 (CCK), 10 nM BRL37344 (BRL), 20 nM insulin (INS), 100 nM dexamethasone (DXM), 10 lM forskolin (FSK) and 10 lM all-trans retinoic acid (ATRA). (C) The cells were treated for 48 h with or without 10 nM BRL37344 (BRL),or forskolin (FSK). 24-h (A, B) or 48-h (C) immunoreactive leptin release in the culture medium was determined by radioimmunoassay of leptin (RIA Kit fom Linco). Data represent means ± S.E.M. for five independent experiments performed in triplicate. \P < 0.05; \\P < 0.01; \\\P < 0.001 vs. control.

Article Snippet: The cells were incubated overnight at 4 C with rabbit polyclonal anti-human leptin antibody diluted at 1:50 (BioVendor GmH, Heidelberg, Germany).

Techniques: RIA Assay, Control

Journal: FEBS letters

Article Title: Similarities and differences in the transcriptional regulation of the leptin gene promoter in gastric and adipose cells.

doi: 10.1016/j.febslet.2005.02.031

Figure Lengend Snippet: Fig. 1. MKN-74 cells expressed leptin and gut hormone receptors. (A) RT-PCR analysis from total RNA isolated from MKN-74 cells. LEP gene: -RT: omission of reverse transcriptase and PCR amplification with ob primers result in a PCR product of 241 bp. CCK-receptors: -RT: omission of reverse transcriptase; CCK1-receptor (CCK-1R): 398 bp PCR product and CCK2-receptor (CCK-2R): a 362 bp PCR product. b3-adrenoceptor. lanes 1 and 2: PCR amplification with human b3 primers result in a PCR product of 525 bp in two separate samples. Insulin receptor: PCR amplification with human insulin receptor primers result in a PCR product of 770 bp. (B) Immunode- tection of leptin protein in human MKN-74 cells. Immunostaining was carried out with a specific antibody for leptin, followed by biotinylated second antibody, streptavidine-peroxydase and diaminobenzidine. Leptin immunoreactivity was visible in the intracytoplasmic compart- ments and on the cell membrane (bar = 20 lm). Note that there is no leptin immunostaining after preabsorption of anti-human leptin rabbit polyclonal antibody with the recombinant human leptin at 10 lg/mL overnight at 4 C. (C) Leptin secretion by gastric MKN-74 cells. Cells were cultured as described in Section 2. The culture medium was sampled at the indicated period of time for leptin determination by radioimmunoassay (Linco Research, St. Charles, MO, USA). Each point represents the mean ± 1S.E.M. of four independent experiments performed in duplicate.

Article Snippet: The cells were incubated overnight at 4 C with rabbit polyclonal anti-human leptin antibody diluted at 1:50 (BioVendor GmH, Heidelberg, Germany).

Techniques: Reverse Transcription Polymerase Chain Reaction, Isolation, Reverse Transcription, Immunostaining, Membrane, Recombinant, Cell Culture, RIA Assay

Journal: FEBS letters

Article Title: Similarities and differences in the transcriptional regulation of the leptin gene promoter in gastric and adipose cells.

doi: 10.1016/j.febslet.2005.02.031

Figure Lengend Snippet: Fig. 3. Hormonal regulation of the human leptin promoter in gastric MKN-74 and in adipose 3T3-L1 cells. Luciferase reporter plasmid containing the leptin promoter or promoterless (basic) was transfected into gastric MKN-74 and differentiated adipose 3T3-L1 cells as described Section 2. The efficiency of the transfections studies monitored by evaluation of the control vector pGL3-Basic activity in comparison to the LEP promoter pGL3-dOb2 showed a 4-fold luciferase expression in MKN-74 and a 1.5-fold in adipose 3T3-L1 cells. The cells were treated with or without 10 nM CCK8 (CCK), 10 nM BRL37344 (BRL), 20 nM insulin (INS), 100 nM dexametha- sone (DXM), 10 lM forskolin (FSK) and 10 lM all-trans retinoic acid (ATRA). Luciferase activity after 24 h was measured as described Section 2 and normalized to dual Luciferase activity as an internal control. The data calculated as ratio and expressed as means ± S.E.M. for five independent experiments performed in triplicate. *P < 0.05; **P < 0.01 vs. control.

Article Snippet: The cells were incubated overnight at 4 C with rabbit polyclonal anti-human leptin antibody diluted at 1:50 (BioVendor GmH, Heidelberg, Germany).

Techniques: Luciferase, Plasmid Preparation, Transfection, Control, Activity Assay, Comparison, Expressing