Journal: Frontiers in Physiology

Article Title: Impact of Bilateral Sympathetic Stellate Ganglionectomy on TGF-β1 Signaling Pathway in Rats With Chronic Volume Overload

doi: 10.3389/fphys.2020.00375

Figure Lengend Snippet: Expressions of norepinephrine (NE) and acetylcholine (ACh) in the plasma, right ventricle (RV), and left ventricle (LV) by ELISA. (A) The expression of NE was significantly increased in the abdominal aorta-cava fistula (AV) group and decreased in the bilateral sympathetic stellate ganglionectomy after abdominal aorta-cava fistula (AD) group in the plasma, RV, and LV. (B) The expression of ACh was significantly decreased in the AV group and increased in the AD group in the RV (* P < 0.05, ** P < 0.01) (three replicate experiments were performed).

Article Snippet: Acetylcholine (ACh) concentrations of the plasma, RV, and LV were determined with the ELISA kit (BioVision, E4454-100, Milpitas, CA, United States) following the manufacturer’s instructions.

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Expressing

Journal: Neurochemical Research

Article Title: Morin Improves Cognitive Deficits in an in Vivo Model of Vascular Dementia by Modulating the N-methyl-D-aspartate Receptor Signaling Pathways

doi: 10.1007/s11064-026-04717-7

Figure Lengend Snippet: Morin modulated the expression of NMDA receptors in the hippocampus of VaD rats. a the expression levels of NR1 ; b the expression levels of NR2A ; c the expression levels of NR2B ; d the expression levels of NR1 protein; e the expression levels of NR2A protein; f the expression levels of NR2B protein; g protein levels of p-CREB; h protein levels of p-CAMK2A; i protein levels of p-CAMK2D. Protein levels of NR1, NR2A, and NR2B were quantified by ELISA. Data are presented as mean ± SD ( n = 8 per group). Statistical analysis was performed by one-way ANOVA with Tukey’s post-hoc test (data met assumptions of normality and homoscedasticity)/Kruskal-Wallis with Dunn’s test (data did not meet assumptions). * indicates a significant difference from the Sham group; # indicates a significant difference 2VO group; *# indicates a significant difference from both the Sham and 2VO groups, with a p-value of less than 0.05 considered statistically significant

Article Snippet: The phosphorylation status of another key NMDAR downstream effector, CREB at Ser133 (p-CREB), was quantified in hippocampal homogenates using a commercial ELISA kit (Phospho-CREB [Ser133] ELISA Kit, Boster Bio, #EKC2382).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Quercetin enhances decidualization through AKT-ERK-p53 signaling and supports a role for senescence in endometriosis

doi: 10.1186/s12958-024-01265-z

Figure Lengend Snippet: Quercetin inhibits eSC proliferation and enhances decidualization. (A) Control endometrial stromal cells (eSCs) were treated with vehicle (Veh) or quercetin (Q, 6.25-50 µM) for 72 h and the relative cell number was determined. Each point represents data from one individual’s eSCs, with the group mean ± SD for vehicle and each Q dose. (B-C) Control eSCs were treated with vehicle (Veh) or quercetin (1.56-50 µM) for 4 h prior to the addition of cAMP alone (B) or cAMP + MPA (C). After 48 h, decidualization was analyzed by measuring IGFBP1 levels by ELISA. Each point represents data from one individual’s eSCs, as IGFBP1 (percent Veh, where 100% = Veh + cAMP alone (IGFBP1) (B) or Veh + cAMP + MPA (IGFBP1) (C) , with the group median ± interquartile range (IQR) for each Q dose. (D) eSCs from two control subjects were treated with vehicle (Veh) or quercetin (Q, 25µM) for 4 h followed by vehicle or cAMP + MPA prior to immunofluorescent staining and confocal imaging; images show IGFBP1 (red), phalloidin (green), and DAPI (blue) staining (at 20x magnification). The scale bars (20 μm) are indicated. IGFBP1 + cells are indicated by yellow arrows. Images showing individual channels are in Supplementary Fig. . ( E – G ) The effects of quercetin (Q, 25 µM) on decidualization when added 4 h pre-cAMP ± MPA ( E ) versus at the same time as (0 h pre-) cAMP ± MPA ( F ) or 20 h post-cAMP ± MPA stimulation ( G ). IGFBP1 levels were measured by ELISA 48 h post cAMP ± MPA stimulation. Data are shown as IGFBP1 (% Veh) where 100% = Veh + cAMP alone (IGFBP1). Each point represents data from one individual’s eSCs, with the median ± IQR shown for each group comparing Veh vs. Q. * p < 0.05, ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns = non-significant

Article Snippet: Intracellular cAMP ([cAMP] i ) production by eSCs isolated from controls and endometriosis cases was analyzed using the cAMP ELISA kit (Cayman Chemical, Ann Arbor, MI, US) according to the manufacturer’s guidelines.

Techniques: Control, Enzyme-linked Immunosorbent Assay, Staining, Imaging

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Quercetin enhances decidualization through AKT-ERK-p53 signaling and supports a role for senescence in endometriosis

doi: 10.1186/s12958-024-01265-z

Figure Lengend Snippet: Endometriosis eSCs exhibit impaired decidualization. (A-B) Comparison of decidualization responses of eSCs from controls (CTRL) vs. endometriosis (ENDO) cases induced by cAMP alone (A) or cAMP + MPA (B) , as determined by IGFBP1 levels measured 48 h post cAMP ± MPA by ELISA. Each point represents data from one individual’s eSCs, with mean ± SD shown for each group. ** p < 0.001 CTRL vs. ENDO (Student’s t test)

Article Snippet: Intracellular cAMP ([cAMP] i ) production by eSCs isolated from controls and endometriosis cases was analyzed using the cAMP ELISA kit (Cayman Chemical, Ann Arbor, MI, US) according to the manufacturer’s guidelines.

Techniques: Comparison, Enzyme-linked Immunosorbent Assay

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Quercetin enhances decidualization through AKT-ERK-p53 signaling and supports a role for senescence in endometriosis

doi: 10.1186/s12958-024-01265-z

Figure Lengend Snippet: Quercetin enhances decidualization by control-eSCs and endometriosis-eSCs, as determined by IGFBP1 and PRL protein production. (A-H) Endometrial stromal cells (eSCs) from controls ( A , C , E , and G , CTRL) and endometriosis cases ( B , D , F , and H , ENDO) were treated with vehicle (Veh) or quercetin (Q, 25µM) for 4 h prior to the addition of cAMP alone ( A - B , E - F ) or cAMP + MPA ( C - D , G - H ). After 48 h, decidualization was analyzed by measuring IGFBP1 ( A - D ) or PRL ( E - H ) levels by ELISA. Data points connected by a line represent paired data points from one individual’s eSCs (± Q). * p < 0.05 Veh vs. Q-treated; ** p < 0.01 Veh vs. Q-treated; *** P < 0.001 Veh vs. Q-treated; **** p < 0.0001 Veh vs. Q-treated

Article Snippet: Intracellular cAMP ([cAMP] i ) production by eSCs isolated from controls and endometriosis cases was analyzed using the cAMP ELISA kit (Cayman Chemical, Ann Arbor, MI, US) according to the manufacturer’s guidelines.

Techniques: Control, Enzyme-linked Immunosorbent Assay

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Quercetin enhances decidualization through AKT-ERK-p53 signaling and supports a role for senescence in endometriosis

doi: 10.1186/s12958-024-01265-z

Figure Lengend Snippet: Quercetin does not increase [cAMP]i concentrations in eSCs. (A-B) Endometrial stromal cells (eSCs) from controls (CTRL) or endometriosis cases (ENDO) were treated with IBMX (0.1mM, a phosphodiesterase inhibitor that blocks [cAMP]i degradation), followed by addition of either vehicle, forskolin (FOR, 25µM, an activator of adenylyl cyclases and hence, [cAMP]i) or quercetin (Q, 25µM). Lysates were analyzed by ELISA for [cAMP]i concentrations (A-B). For clarity, data without forskolin (FOR) treatment are also shown on a different scale (B). Each point represents data from one individual’s eSCs, with median ± IQR shown for each group. ** p < 0.001 vs. vehicle (IBMX alone); ns = not significant

Article Snippet: Intracellular cAMP ([cAMP] i ) production by eSCs isolated from controls and endometriosis cases was analyzed using the cAMP ELISA kit (Cayman Chemical, Ann Arbor, MI, US) according to the manufacturer’s guidelines.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Quercetin enhances decidualization through AKT-ERK-p53 signaling and supports a role for senescence in endometriosis

doi: 10.1186/s12958-024-01265-z

Figure Lengend Snippet: Quercetin inhibits AKT and ERK1/2 phosphorylation and signaling and promotes p53 (Ser46) phosphorylation. (A-B) Control endometrial stromal cells (eSCs) were treated with quercetin (Q, 25µM) for 4 h before analyzing cell lysates using a phospho-kinase array panel. Representative arrays (A) and quantification of array analytes/spots (B) are shown. Data for differentially expressed targets are presented as specific spot density normalized to the reference spot comparing Veh- vs. Q-treated (B). Each point represents the specific density of each duplicate analyte spot as a percentage of Veh-treated, with group median ± IQR shown for control- and endometriosis-eSCs. ** p < 0.01. The remaining arrays are in Supplementary Fig. . ( C - D ) Control (CTRL) and endometriosis (ENDO) eSCs were treated with Veh or Q (25µM) for 4 h before western blotting for p-AKT, total AKT, p-ERK1/2, total ERK1/2, p-PRAS40, and total PRAS40. Representative blots and quantification of specific analytes are shown in (C) and (D) , respectively. Band densities were normalized to GAPDH and shown as fold-change between Veh- vs. Q-treated eSCs in (D). Each point represents data from one individual’s eSCs, with median ± IQR shown for each group. * p < 0.05; *** p < 0.001; **** p < 0.0001. (E) Control-eSCs were treated with Veh or Q (25µM) for 0.5, 2, and 4 h before western blotting for phospho-AKT, total AKT, phospho-ERK1/2, total ERK1, phospho-PRAS40, and total PRAS40. (F-I) Control-eSCs were treated with vehicle (Veh), AKT inhibitor MK-2206 (MK, 1µM), quercetin (Q, 25µM) or Q + MK for 4 h prior to cAMP (F and H) or cAMP + MPA (G and I) stimulation; decidualization was assessed by measuring IGFBP1 (F and G) or PRL (H and I) by ELISA. Data are presented as fold-change in IGFBP1 or PRL over Veh-treated (Veh-treated = 1). Each symbol represents data from one individual’s eSCs, with median ± IQR shown for each group. * p < 0.05; ** p < 0.01 vs. Veh-treated; *** p < 0.001 vs. Veh-treated; ns = non-significant vs. Q-treated

Article Snippet: Intracellular cAMP ([cAMP] i ) production by eSCs isolated from controls and endometriosis cases was analyzed using the cAMP ELISA kit (Cayman Chemical, Ann Arbor, MI, US) according to the manufacturer’s guidelines.

Techniques: Phospho-proteomics, Control, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Quercetin enhances decidualization through AKT-ERK-p53 signaling and supports a role for senescence in endometriosis

doi: 10.1186/s12958-024-01265-z

Figure Lengend Snippet: eSCs show evidence of senescence-like phenotype, which is reversed by quercetin. (A) Endometriosis (ENDO)-endometrial stromal cells (eSCs) and control (CTRL)-eSCs were treated with vehicle or quercetin (Q, 25 µM) for 2 days before western blotting for SASPs, IL-6 and MMP3, or GAPDH. A representative blot is shown. (B-C) Quantification of blots (with band densities normalized to GAPDH) from control-eSCs treated with vehicle (Veh) or quercetin (Q, 25 µM) for 2 days for IL-6 and MMP3 are shown in (B) and (C) , respectively. Data are presented as IL-6% vehicle or MMP3% vehicle (where Veh-treated = 100%). Each point represents data from one individual’s eSCs, with median ± IQR shown for each group * p < 0.05; ** p < 0.01; ns = non-significant. (D) Control-eSCs were treated with vehicle or 250 µM H2O2 for 2 h and then, eSCs were harvested at 2, 4, 6, and 8 days post-H2O2 exposure for western blotting analysis for p21, p16, lamin B1, MMP3, and GAPDH. (E) Control-eSCs were treated with vehicle (Veh) or H2O2 (250 µM) for 2 h and then treated with vehicle (Veh) or quercetin (Q, 25 µM), as indicated, for 4 h prior to cAMP + MPA-induced decidualization. After 48 h, culture supernatants were assessed for IGFBP1 (pg/ml) by ELISA. Data points connected by lines represent paired data points from one individual’s eSCs (comparing Veh vs. H2O2 and H2O2-Q vs. H2O2 + Q). * p < 0.05; ** p < 0.01 Veh vs. Q-treated; ns = non-significant

Article Snippet: Intracellular cAMP ([cAMP] i ) production by eSCs isolated from controls and endometriosis cases was analyzed using the cAMP ELISA kit (Cayman Chemical, Ann Arbor, MI, US) according to the manufacturer’s guidelines.

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

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Quercetin enhances decidualization through AKT-ERK-p53 signaling and supports a role for senescence in endometriosis

doi: 10.1186/s12958-024-01265-z

Figure Lengend Snippet: Inhibiting apoptosis blocks decidualization and quercetin induces apoptosis in a subset of eSCs. (A-B) Control-eSCs (p3) were treated with vehicle (Veh) or Z-VAD-fmk (0 vs. 40 µM), a pan-caspase inhibitor that blocks apoptosis, in the presence of vehicle (A) or quercetin (Q, 25 µM) (B) prior to inducing decidualization with cAMP + MPA. Decidualization was assessed 48 h later by ELISA for IGFBP1. Each point represents data from one individual’s eSCs, with the median ± IQR shown for each group. ** p < 0.01. (C-D) Quercetin induces apoptosis in a subset of control-eSCs (passage 3–4), as determined by flow cytometry. Representative gating (upper panels) and histograms (lower panels) of Annexin V staining comparing vehicle-treated (Veh, left panels) vs. quercetin-treated (Q, 25 µM, right panels) eSCs 24 h post-treatment (C). Quantification of quercetin-induced apoptosis (shown as fold-change) measured 24 h and 48 h post-vehicle (Veh) vs. post-quercetin (Q, 25 µM) treatment (D). Each point represents data from one individual’s eSCs (fold-change in apoptosis), with median ± IQR shown for each group (± Q). ** p < 0.01. (E-F) Larger senescence-associated β-galactosidase (SA-βgal) + control-eSCs are reduced following quercetin treatment. SA-βgal was determined using 9 H-(1,3-Dichloro-9,9-Dimethylacridin-2-one) β-D-Galactopyranoside (DDAOG), a substrate of SA-βgal that yields a far-red fluorescent product detected by flow cytometry at 670 nm. Y-axis (FSC-A, size) and X-axis (DDAOG, 670 nm). Representative flow cytometry plots show the gating of larger DDAOG, 670 nm/SA-βgal (Beta-gal) + eSCs comparing vehicle-treated (Veh – left panel) vs. quercetin-treated (Q, 25 µM- right panel) eSCs (p3-4) at 48 h post-treatment (E). The percentage of Beta-gal+/senescent cells (of total viable cells per eSC sample) is indicated on the representative plots in E. The fold-change in the number of larger Beta-gal+/senescent cells comparing vehicle (Veh) vs. quercetin (Q, 25 µM) is shown (F). Each point represents data from one individual’s eSCs (fold-change in larger Beta-gal + cells), with median ± IQR shown for each group (± Q). * p < 0.05 comparing vehicle vs. Q

Article Snippet: Intracellular cAMP ([cAMP] i ) production by eSCs isolated from controls and endometriosis cases was analyzed using the cAMP ELISA kit (Cayman Chemical, Ann Arbor, MI, US) according to the manufacturer’s guidelines.

Techniques: Control, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Staining