Journal: Biology of reproduction

Article Title: 17Beta-estradiol enhances leptin expression in human placental cells through genomic and nongenomic actions.

doi: 10.1095/biolreprod.110.083535

Figure Lengend Snippet: FIG. 1. E2 enhances leptin expression in placental cells. A) BeWo cells were incu- bated for 72 h with different E2 doses, as indicated, in DMEM-F12 media supple- mented with 1% charcoal-stripped FCS. B) Placental explants were processed as de- scribed in Materials and Methods and treated with different E2 doses for 4 h as indicated. C) BeWo cells were incubated for 24 (lanes 1–3), 48 (lanes 4–6), or 72 h (lanes 7–9) and treated with or without 10 and 100 nM E2. D) Placental explants were incu- bated with or without different doses of the antiestrogen ICI 182 780 for 4 h, in the presence or absence of 100 nM E2. In all cases, cell extracts were prepared as indi- cated in Materials and Methods. Proteins were separated on SDS-PAGE gels, and leptin expression was determined by West- ern blot. Loading controls were performed by immunoblotting the same membranes with anti-ACTB. Band densitometry is shown in lower panels. Representative results from three replicates are shown. Statistical analysis was performed by AN- OVA. Significant differences from control were determined by Dunnett multiple comparison post hoc test. *P , 0.05; #P , 0.01; zP , 0.001 vs. control. a.u., arbitrary units.

Article Snippet: Membranes were equilibrated in 13 PBS, and nonspecific binding sites were blocked by 5% nonfat milk in PBS at room temperature for 1 h. Membranes were then immunoblotted with polyclonal rabbit anti-human leptin Y20 (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA) with polyclonal rabbit anti-phospho-mitogen-activated protein kinases 1 and 3 (MAPK1/3; Thr202/Tyr204; 1:3000) or with polyclonal rabbit anti-phospho-AKT (Ser 472/473) antibody (1:3000; BD Biosciences Pharmingen, NJ).

Techniques: Expressing, Incubation, SDS Page, Western Blot, Control, Comparison

Journal: Biology of reproduction

Article Title: 17Beta-estradiol enhances leptin expression in human placental cells through genomic and nongenomic actions.

doi: 10.1095/biolreprod.110.083535

Figure Lengend Snippet: FIG. 2. E2 enhances leptin promoter activity in BeWo cells. A) Cells were transiently transfected with pL1951 plasmid construction and treated with E2 as indicated. B) Cells were transfected with pL1951 and incubated with different doses of ICI 182 780, in the presence or absence of 100 nM E2. C) Cells were transiently transfected with plasmids containing different leptin promoter deletions and treated with (black bars) or without (white bars) 100 nM E2. D) Cells were transfected with plasmids derived from pGL3 promoter containing fragments of leptin promoter upstream of SV40 promoter and incubated with 10 nM E2 (gray bars), 100 nM E2 (black bars), or not treated (white bars). Numbers at right show basal promoter activity of each construct relative to the empty vector. In all cases, after transfection, cells were incubated for 48 h in DMEM-F12 media supplemented with 1% charcoal-stripped FCS. Luciferase activity was measured in cellular extracts and normalized to b-galactosidase activity. Activity obtained in the absence of E2 and ICI 182 780 was set as control. Results are expressed as mean 6 SD for three independent experiments performed in duplicate. Statistical analysis was performed by ANOVA. Significant differences from control were determined by Dunnett multiple comparison post hoc test. *P , 0.05; #P , 0.01; zP , 0.001 vs. control. a.u., arbitrary units.

Article Snippet: Membranes were equilibrated in 13 PBS, and nonspecific binding sites were blocked by 5% nonfat milk in PBS at room temperature for 1 h. Membranes were then immunoblotted with polyclonal rabbit anti-human leptin Y20 (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA) with polyclonal rabbit anti-phospho-mitogen-activated protein kinases 1 and 3 (MAPK1/3; Thr202/Tyr204; 1:3000) or with polyclonal rabbit anti-phospho-AKT (Ser 472/473) antibody (1:3000; BD Biosciences Pharmingen, NJ).

Techniques: Activity Assay, Transfection, Plasmid Preparation, Incubation, Derivative Assay, Construct, Luciferase, Control, Comparison

Journal: Biology of reproduction

Article Title: 17Beta-estradiol enhances leptin expression in human placental cells through genomic and nongenomic actions.

doi: 10.1095/biolreprod.110.083535

Figure Lengend Snippet: FIG. 4. MAPK1/3 and AKT are activated by E2 and are involved in placental leptin expression. A, B) Placental explants were processed as previously described and treated with E2 for 10 min. MAPK1/3 (A) or AKT (B) phosphorylation was determined by Western blot as indicated in Materials and Methods. Total MAPK or AKT protein levels in cell extracts were determined as loading controls. C) Cells were transfected with pL1951 and vectors encoding the dominant negative mutants for MAPK1 and MAP2K1 (MAPK-kd and MAP2K-kd, respectively). BeWo cells were incubated for 48 h with 100 nM of E2 (black bars) or without (white bars). Cell extracts were prepared as indicated in Materials and Methods, and luciferase activity was normalized to b-galactosidase activity. Percentage of inhibition is shown above bars. #P , 0.01 vs. control. D and E) BeWo cells were pretreated with or without PD98059 and Wortmannin for 20 min and then were incubated for 48 h with E2 (D), E-BSA (E), or without them. Leptin expression was determined by Western blot. In Western blot analysis, samples were loaded in a 12% SDS-PAGE. Band densitometry is shown in the lower panel. Results from a representative experiment are shown. They are expressed as mean 6 SD of two independent experiments. Significant differences from control were determined by Dunnett multiple comparison post hoc test. #P , 0.01 vs. control. a.u., arbitrary units.

Article Snippet: Membranes were equilibrated in 13 PBS, and nonspecific binding sites were blocked by 5% nonfat milk in PBS at room temperature for 1 h. Membranes were then immunoblotted with polyclonal rabbit anti-human leptin Y20 (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA) with polyclonal rabbit anti-phospho-mitogen-activated protein kinases 1 and 3 (MAPK1/3; Thr202/Tyr204; 1:3000) or with polyclonal rabbit anti-phospho-AKT (Ser 472/473) antibody (1:3000; BD Biosciences Pharmingen, NJ).

Techniques: Expressing, Phospho-proteomics, Western Blot, Transfection, Dominant Negative Mutation, Incubation, Luciferase, Activity Assay, Inhibition, Control, SDS Page, Comparison