Journal: Frontiers in Endocrinology

Article Title: The effects of leptin on human cytotrophoblast invasion are gestational age and dose-dependent

doi: 10.3389/fendo.2024.1386309

Figure Lengend Snippet: Leptin has a differential effect on HTR-8/SVneo and second-trimester human pCTB invasion. (A) Invasion of HTR-8/Svneo cells through Matrigel-coated invasion chambers was evaluated in the presence of 0, 10, 80, 160 or 320 ng/ml leptin. Data normalized to invasion at 0 ng/ml leptin are combined from three independent experiments: n-12, 8, 14, 13, and 12 invasion chambers per respective leptin concentration. (B) Absolute cell number of HTR-8/Svneo cells cultured on Matrigel in the presence of 0-320 ng/ml leptin was evaluated. Number of cells after 48 hours was normalized to the number of cells plated at the start of the experiment. Normalized data from three independent experiments were combined, n-12 wells per concentration. (C) Invasion of pCTBs plated as described for (A) Primary CTBs isolated from six placentas (13-24 weeks gestation) were evaluated independently and normalized data combined; n=20, 19, 19, 13, 7 invasion chambers per respective leptin concentration. Data are reported as mean fold change from 0 ng/ml +/- standard error of mean (SEM). *p<0.05 vs. 0 ng/ml, **p<0.01 vs. 0 ng/ml, ^p<0.05 vs. 10 ng/ml, # p<0.01 vs. 80 ng/ml.

Article Snippet: Invasion medium [HTR-8/SVneo: RPMI 1640. pCTBs: DMEM high glucose (Hyclone) supplemented with 2% Nutridoma-SP (Roche) and 50 µg/ml gentamycin (Gibco)] containing 0, 10, 80, 160, or 320 ng/ml human recombinant leptin (Sigma) was added in both the upper chamber and the bottom well.

Techniques: Concentration Assay, Cell Culture, Isolation

Journal: Frontiers in Endocrinology

Article Title: The effects of leptin on human cytotrophoblast invasion are gestational age and dose-dependent

doi: 10.3389/fendo.2024.1386309

Figure Lengend Snippet: Leptin affects human explant CTB invasion in a dose and gestational age-dependent manner. Invasion of placental villous explants plated on Matrigel was evaluated in the presence of 0-320 ng/ml leptin. The area of Matrigel invaded by CTBs after three days of leptin treatment (Day 3) is normalized to the area invaded on Day 0, before the initiation of leptin treatment. Normalized data are combined from independent experiments. (A) A representative image of the CTB invasion on Days 0 and 3. (B) Fold change invasion of <8 weeks gestation explant CTBs increases with increasing leptin dose (5- and 7-week gestation placentas; n=6-8 explants per leptin concentration). (C) Fold change invasion of 8-10-week gestation explant CTBs is not differentially affected by leptin (8, 9, and 10-week gestation placentas; n=8-14 explants per leptin concentration). (D) Fold change invasion of explant CTBS from two independent 11-week gestations had a differentially invasive trend in response to leptin (n=5-6 explants per leptin concentration). Data are reported as mean fold change in the area invaded on Day 3 compared to Day 0 +/- SE. *p<0.05 vs. 0 ng/ml, ^p<0.05 vs. 10 ng/ml.

Article Snippet: Invasion medium [HTR-8/SVneo: RPMI 1640. pCTBs: DMEM high glucose (Hyclone) supplemented with 2% Nutridoma-SP (Roche) and 50 µg/ml gentamycin (Gibco)] containing 0, 10, 80, 160, or 320 ng/ml human recombinant leptin (Sigma) was added in both the upper chamber and the bottom well.

Techniques: Concentration Assay

Journal: Frontiers in Endocrinology

Article Title: The effects of leptin on human cytotrophoblast invasion are gestational age and dose-dependent

doi: 10.3389/fendo.2024.1386309

Figure Lengend Snippet: Leptin did not change MMP-2 or MMP-9 activity in human CTBs cultured on Matrigel. Culture medium from the top chamber of the invasion assays was collected and analyzed for MMP-2 and -9 activity by gelatin zymography. Representative images are shown from the conditioned medium after 24 hours for 17-week pCTBs and HTR-8/Svneo cells (conditioned medium from n=3 invasion assays). Similar results were seen for pCTB regardless of gestational age (n=4, 17-23 weeks gestation). Serum-free medium served as the negative control (-), and medium containing serum served as the positive control (+).

Article Snippet: Invasion medium [HTR-8/SVneo: RPMI 1640. pCTBs: DMEM high glucose (Hyclone) supplemented with 2% Nutridoma-SP (Roche) and 50 µg/ml gentamycin (Gibco)] containing 0, 10, 80, 160, or 320 ng/ml human recombinant leptin (Sigma) was added in both the upper chamber and the bottom well.

Techniques: Activity Assay, Cell Culture, Zymography, Negative Control, Positive Control

Journal: Frontiers in Endocrinology

Article Title: The effects of leptin on human cytotrophoblast invasion are gestational age and dose-dependent

doi: 10.3389/fendo.2024.1386309

Figure Lengend Snippet: Leptin did not change LepR expression in HTR8/SVneo cells. HTR8/SVneo cells were treated with indicated concentration of leptin and total RNA was isolated 24 hours (Upper panel) and 48 hours (Lower panel) hours post-treatment. Total RNA was used to perform qPCR to determine mRNA levels of Leptin receptor using pan Lep R primers (A, B) and isoform specific primers (C, D) . Bar graphs represent the mean fold change relative to the control (Ong/ml). The gene expression values for LepR at each dose were normalized to 18S. Data are depicted as Mean ± SEM from three independent experiments.

Article Snippet: Invasion medium [HTR-8/SVneo: RPMI 1640. pCTBs: DMEM high glucose (Hyclone) supplemented with 2% Nutridoma-SP (Roche) and 50 µg/ml gentamycin (Gibco)] containing 0, 10, 80, 160, or 320 ng/ml human recombinant leptin (Sigma) was added in both the upper chamber and the bottom well.

Techniques: Expressing, Concentration Assay, Isolation

Journal: Frontiers in Endocrinology

Article Title: The effects of leptin on human cytotrophoblast invasion are gestational age and dose-dependent

doi: 10.3389/fendo.2024.1386309

Figure Lengend Snippet: Alterations in STAT, PI3K, and MAPK signaling protein phosphorylation were evident in response to leptin, as determined by a phosphokinase array. HTR-8/SVneo cells, cultured on matrigel for an hour were treated with 0, 80, or 320ng/ml leptin for 30 minutes and were subjected to phosphokinase array analysis. Out of 46 unique phosphorylation sites evaluated, 16 sites showed greater than a 1.2-fold (20%) difference in phosphorylation between 0, 80, and 320ng/ml leptin. The dotted line represents a threshold of 1.2- fold. Leptin doses 80 and 320ng/ml are shown as gray and black bars respectively. Changes in phosphorylation in (A) STAT pathway, (B) PI3K pathway (C) MAPK pathway, and (D) Integrin and Cell Cycle Pathways. STAT, PI3K, and MAPK signaling may be involved in the differential invasive response to leptin.

Article Snippet: Invasion medium [HTR-8/SVneo: RPMI 1640. pCTBs: DMEM high glucose (Hyclone) supplemented with 2% Nutridoma-SP (Roche) and 50 µg/ml gentamycin (Gibco)] containing 0, 10, 80, 160, or 320 ng/ml human recombinant leptin (Sigma) was added in both the upper chamber and the bottom well.

Techniques: Cell Culture

Journal: Cell reports

Article Title: Lung Mammary Metastases but Not Primary Tumors Induce Accumulation of Atypical Large Platelets and Their Chemokine Expression

doi: 10.1016/j.celrep.2019.10.016

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Goat anti-hamster, AF488 , Jackson ImmunoResearch , Cat# 127-545-160, RRID:AB_2338997.

Techniques: Recombinant, RNA Sequencing, Software