Journal: Scientific Reports

Article Title: Blocking Endogenous Leukemia Inhibitory Factor During Placental Development in Mice Leads to Abnormal Placentation and Pregnancy Loss

doi: 10.1038/srep13237

Figure Lengend Snippet: Wild type (WT) mouse implantation sites were collected from n = 3 mice/timepoint. ( a ) LIF, ( b ) LIFRα and ( c ) gp130 mRNA expression were analysed in E10, 13, 15 and 17 placenta by quantitative real-time PCR, normalized to β2-microglobulin. Data are mean ± SEM, ANOVA, Tukey’s post-hoc test, *p < 0.05, **p < 0.01, ***p < 0.001. ( d ) LIF protein expression was analysed in E6, 8, 10 implantation sites and E13, 15 and 17 placenta by Western blot, normalized to GAPDH. ( e ) Data are mean ± SEM, ANOVA, Tukey’s post-hoc test, *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: For immunohistochemistry, sections were microwaved at high power (700 W) in 0.01 M citric acid buffer (pH 6.0) for 5 min. Endogenous peroxidase activity was quenched with 6% H 2 O 2 in 100% methanol (1:1 v/v) for 10 min. Tissues were incubated with non-immune blocking solution (10% normal horse serum, 2% normal mouse serum) diluted in 1 ×Tris-buffered saline (TBS) for 30 min. Primary antibody for LIFRα (1:100; R&D Systems #AF-249NA), LIF (1:100; R&D Systems #AF-250NA), phospho-STAT3 (Tyr705) (1:100; Cell Signaling Technologies #9145), phospho-p44/42 MAPK (Erk1/2, Thr202/Tyr204) (1:100; Cell Signaling Technologies #4370), pan-cytokeratin (1:200; Santa Cruz #H-240), CD31 (1:100; Abcam #ab28364), desmin (1:100; Dako, #D-33), isolectin-B4 (ISB4) (5 μg/section; Sigma #L5391) or non-immune goat IgG (isotype negative control) in blocking solution were applied for 18 h and incubated at 4 °C.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot

Journal: Scientific Reports

Article Title: Blocking Endogenous Leukemia Inhibitory Factor During Placental Development in Mice Leads to Abnormal Placentation and Pregnancy Loss

doi: 10.1038/srep13237

Figure Lengend Snippet: Wild type (WT) mouse implantation sites were collected from n = 3 mice/timepoint and immunostained for LIF and LIFRα. Representative photomicrographs of mid-gestation (E13) implantation site sections are shown here. ( a ) LIF localized to decidual cells, ( b ) trophoblast giant cells, ( c ) spongiotrophoblast islands (line) and ( d ) some mononuclear trophoblasts associated with maternal vascular spaces (arrow) as well as endothelial cells lining fetal cappillaries (arrow head). ( e ) LIFRα was produced abundantly throughout the implantation sites. ( f ) LIFRα localized to decidual cells. In the junctional zone LIFRα was produced by ( g ) trophoblast giant cells, ( h ) spongiotrophoblast islands (line) and opposing glycogen cells (arrows). In the placental labyrinth, LIFRα localized to ( i ) syncytiotrophoblasts and mononuclear trophoblasts associated with maternal vascular spaces and also ( j ) endothelial cells lining fetal cappillaries (arrow heads). Bars represent 20 μm ( a–d, f–j ) and 200 μm ( e ). Insets are negative controls.

Article Snippet: For immunohistochemistry, sections were microwaved at high power (700 W) in 0.01 M citric acid buffer (pH 6.0) for 5 min. Endogenous peroxidase activity was quenched with 6% H 2 O 2 in 100% methanol (1:1 v/v) for 10 min. Tissues were incubated with non-immune blocking solution (10% normal horse serum, 2% normal mouse serum) diluted in 1 ×Tris-buffered saline (TBS) for 30 min. Primary antibody for LIFRα (1:100; R&D Systems #AF-249NA), LIF (1:100; R&D Systems #AF-250NA), phospho-STAT3 (Tyr705) (1:100; Cell Signaling Technologies #9145), phospho-p44/42 MAPK (Erk1/2, Thr202/Tyr204) (1:100; Cell Signaling Technologies #4370), pan-cytokeratin (1:200; Santa Cruz #H-240), CD31 (1:100; Abcam #ab28364), desmin (1:100; Dako, #D-33), isolectin-B4 (ISB4) (5 μg/section; Sigma #L5391) or non-immune goat IgG (isotype negative control) in blocking solution were applied for 18 h and incubated at 4 °C.

Techniques: Produced

Journal: Reproductive Sciences

Article Title: Ulipristal Acetate Mediates Decreased Proteoglycan Expression Through Regulation of Nuclear Factor of Activated T-Cells (NFAT5)

doi: 10.1177/1933719118816836

Figure Lengend Snippet: Ulipristal acetate regulates solute transporter proteins in 3D myometrial and leiomyoma cells. A, Western blot analysis of myometrial cells exposed to UPA show only a slight increase in AKR1B1 protein at the lowest concentration of 10−10 M (1.34 ± 0.08-fold) when compared to placebo myometrial cells. Higher concentrations of UPA show no change in AKR1B1 expression in these cells from placebo controls. B, Western blot analysis of leiomyoma cells treated with UPA show a decrease in the expression of AKR1B1 at all concentrations: 10−10 M (0.72 ± 0.07-fold), 10−9 M (0.63 ± 0.09-fold), and 10−8 M (0.69 ± 0.07-fold) evaluated. C, Western blot analysis of 3D leiomyoma cells demonstrate a decrease in the expression of SLC5A3 protein at 10−8 M (0.74 ± 0.01), 10−7 M (0.66 ± 0.02), and 10−6 M (0.67 ± 0.05) compared to placebo myometrium, when exposed to UPA treatment (*P < .05). D, Western blot analysis of leiomyoma cells treated with UPA demonstrate a maximally 1.34-fold decrease in the expression of SLC5A3 protein for all concentrations of UPA: 10−10 M (0.80 ± 0.12), 10−9 M (0.84 ± 0.01), 10−8 M (0.83 ± 0.12), and 10−7 M (0.74 ± 0.02) evaluated, that was not concentration dependent. AKR1B1 indicates aldo-keto reductase family 1 member B1; SLC5A3, solute carrier family 5 member 3; UPA, ulipristal acetate.

Article Snippet: After incubation in blocking solution (5% nonfat milk in 0.1% Tween 20 in 1× TBS), membranes were washed and exposed to primary antibody against BCAN (sc-166951; 1:400; Santa Cruz Biotechnology), B3GAT1 (1:500; sc-390475), aldo-keto reductase family 1 member B1 (AKR1B1; ab62795; 1:500), solute carrier family 5 member 3 (SLC5A3; ab113245; 1:500; Abcam; ), VCAN (ab28671; 1:500) overnight at 4°C.

Techniques: Western Blot, Concentration Assay, Expressing