Journal: International Journal of Molecular Sciences

Article Title: ASC-Derived Extracellular Vesicles Suppress Macrophage-Driven Inflammatory Amplification and Contractile Activation of Uterine Smooth Muscle Cells

doi: 10.3390/ijms27104273

Figure Lengend Snippet: Characterization and cellular localization of ASC-derived EVs. ( A ) Transmission electron microscopy (TEM) images showing vesicles with sizes consistent with extracellular vesicles (Scale bars = 100 nm). ( B ) Size distribution of EVs measured by nanoparticle tracking analysis (NTA), with a median diameter of 183.1 nm and a particle concentration of 3.8 × 10 10 particles/mL. ( C ) Western blot analysis confirming the expression of EV-associated markers CD9 and CD63. ( D ) Fluorescence microscopy images showing PKH67-labeled EV signals (green) in HUtSMCs. Nuclei were stained with DAPI (blue). Scale bars = 100 mm.

Article Snippet: Human uterine smooth muscle cells (HUtSMCs) were obtained from PromoCell (C-12575, Heidelberg, Germany) and cultured in Smooth Muscle Cell Growth Medium 2 (C-22062, PromoCell (Heidelberg, Germany)) at 37 °C in a humidified atmosphere with 5% CO 2 .

Techniques: Derivative Assay, Transmission Assay, Electron Microscopy, Concentration Assay, Western Blot, Expressing, Fluorescence, Microscopy, Labeling, Staining

Journal: International Journal of Molecular Sciences

Article Title: ASC-Derived Extracellular Vesicles Suppress Macrophage-Driven Inflammatory Amplification and Contractile Activation of Uterine Smooth Muscle Cells

doi: 10.3390/ijms27104273

Figure Lengend Snippet: ASC-derived EVs suppress LPS-induced inflammatory signaling in HUtSMCs. ( A ) HUtSMCs were pretreated with ASC-derived EVs and stimulated with LPS for the indicated time points (5–20 min). Phosphorylation of JNK and ERK1/2 was analyzed by Western blotting. ( B ) mRNA expression levels of TSG6, IL-6, IL-8, MCP-1, and MIP-2 were measured by quantitative RT-PCR at 8–24 h after LPS stimulation and normalized to GAPDH. ( C ) Secretion levels of IL-6, IL-8, and MCP-1 were quantified by ELISA. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ns, not significant.

Article Snippet: Human uterine smooth muscle cells (HUtSMCs) were obtained from PromoCell (C-12575, Heidelberg, Germany) and cultured in Smooth Muscle Cell Growth Medium 2 (C-22062, PromoCell (Heidelberg, Germany)) at 37 °C in a humidified atmosphere with 5% CO 2 .

Techniques: Derivative Assay, Phospho-proteomics, Western Blot, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: ASC-Derived Extracellular Vesicles Suppress Macrophage-Driven Inflammatory Amplification and Contractile Activation of Uterine Smooth Muscle Cells

doi: 10.3390/ijms27104273

Figure Lengend Snippet: ASC-derived EVs modulate TLR4-dependent inflammatory signaling in HUtSMCs. ( A ) HUtSMCs were pretreated with ASC-EVs and/or a TLR4 inhibitor prior to LPS stimulation. Phosphorylation of JNK and ERK1/2 was analyzed by Western blotting. Representative images from three independent experiments are shown. ( B ) mRNA expression levels of IL-6, IL-8, MCP-1, TNF-α, MIP-1α, and MIP-2 were analyzed by quantitative RT-PCR and normalized to GAPDH. Data are presented as mean ± SEM from three independent experiments and are expressed relative to the untreated control. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ns, not significant.

Article Snippet: Human uterine smooth muscle cells (HUtSMCs) were obtained from PromoCell (C-12575, Heidelberg, Germany) and cultured in Smooth Muscle Cell Growth Medium 2 (C-22062, PromoCell (Heidelberg, Germany)) at 37 °C in a humidified atmosphere with 5% CO 2 .

Techniques: Derivative Assay, Phospho-proteomics, Western Blot, Expressing, Quantitative RT-PCR, Control

Journal: International Journal of Molecular Sciences

Article Title: ASC-Derived Extracellular Vesicles Suppress Macrophage-Driven Inflammatory Amplification and Contractile Activation of Uterine Smooth Muscle Cells

doi: 10.3390/ijms27104273

Figure Lengend Snippet: ASC-derived EVs attenuate macrophage-mediated inflammatory signaling in HUtSMCs. ( A ) HUtSMCs were pretreated with ASC-derived EVs and stimulated with conditioned media (CM) derived from RAW264.7 macrophages treated with LPS (10–100 ng/mL). Cells were harvested after 10 min, and phosphorylation of JNK and ERK1/2 was analyzed by Western blotting. ( B ) mRNA expression levels of IL-6, IL-8, MCP-1, TNF-α, MIP-1α, and MIP-2 were analyzed by quantitative RT-PCR 20 h after CM stimulation. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ns, not significant.

Article Snippet: Human uterine smooth muscle cells (HUtSMCs) were obtained from PromoCell (C-12575, Heidelberg, Germany) and cultured in Smooth Muscle Cell Growth Medium 2 (C-22062, PromoCell (Heidelberg, Germany)) at 37 °C in a humidified atmosphere with 5% CO 2 .

Techniques: Derivative Assay, Phospho-proteomics, Western Blot, Expressing, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: ASC-Derived Extracellular Vesicles Suppress Macrophage-Driven Inflammatory Amplification and Contractile Activation of Uterine Smooth Muscle Cells

doi: 10.3390/ijms27104273

Figure Lengend Snippet: ASC-derived EVs inhibit macrophage TLR4-dependent NF-κB and MAPK activation in HUtSMCs. ( A ) RAW264.7 macrophages were stimulated with LPS in the presence or absence of the TLR4 inhibitor TAK242. mRNA expression levels of IL-1β, IL-10, TNF-α, MIP-2, and MCP-1 were analyzed by quantitative RT-PCR and normalized to β-actin. ( B ) HUtSMCs were treated with conditioned media derived from RAW264.7 macrophages under the indicated conditions, with or without ASC-EVs pretreatment. Phosphorylation of p65, JNK, and ERK1/2 was analyzed by Western blotting. ( C ) Immunofluorescence analysis of phosphorylated p65 (green) in HUtSMCs. Nuclei were stained with DAPI (blue). ( D ) Cytoplasmic and nuclear fractionation analysis of NF-κB signaling. Levels of phosphorylated p65, total p65, phosphorylated IκB, and total IκB were analyzed by Western blotting. α-tubulin and Lamin B1 were used as cytoplasmic and nuclear markers, respectively. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ns, not significant.

Article Snippet: Human uterine smooth muscle cells (HUtSMCs) were obtained from PromoCell (C-12575, Heidelberg, Germany) and cultured in Smooth Muscle Cell Growth Medium 2 (C-22062, PromoCell (Heidelberg, Germany)) at 37 °C in a humidified atmosphere with 5% CO 2 .

Techniques: Derivative Assay, Activation Assay, Expressing, Quantitative RT-PCR, Phospho-proteomics, Western Blot, Immunofluorescence, Staining, Fractionation

Journal: International Journal of Molecular Sciences

Article Title: ASC-Derived Extracellular Vesicles Suppress Macrophage-Driven Inflammatory Amplification and Contractile Activation of Uterine Smooth Muscle Cells

doi: 10.3390/ijms27104273

Figure Lengend Snippet: ASC-derived EVs attenuate macrophage-mediated calcium signaling and contractile activity in HUtSMCs. ( A ) Intracellular calcium flux was measured in HUtSMCs following stimulation with conditioned media derived from RAW264.7 macrophages under the indicated conditions. Relative fluorescence intensity was quantified. ( B ) Collagen gel contraction assay of HUtSMCs treated with macrophage-conditioned media with or without ASC-EVs pretreatment. Representative images and quantitative analysis of gel contraction are shown. Blebbistatin (BDM) was used as a positive control. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ns, not significant.

Article Snippet: Human uterine smooth muscle cells (HUtSMCs) were obtained from PromoCell (C-12575, Heidelberg, Germany) and cultured in Smooth Muscle Cell Growth Medium 2 (C-22062, PromoCell (Heidelberg, Germany)) at 37 °C in a humidified atmosphere with 5% CO 2 .

Techniques: Derivative Assay, Activity Assay, Fluorescence, Collagen Gel Contraction Assay, Positive Control

Journal: Mediators of Inflammation

Article Title: Aster spathulifolius Maxim. Alleviates Primary Dysmenorrhea in a Mouse Model by Modulating Myometrial Contractions via NF-κB/COX-2 Pathway Inhibition

doi: 10.1155/mi/1654087

Figure Lengend Snippet: Inhibitory effect of PDR97 on NF-κB activation in HUtSMCs. (A) NF-κB luciferase activity. HUtSMCs were pretreated with PDR97 for 30 min, followed by coincubation with PGF2α (10 µM) and IL-1β (10 ng/mL) for 16 h. (B) Translocation of NF-κB from the cytoplasm to the nucleus. HUtSMCs were pretreated with PDR97 for 30 min, followed by coincubation with PGF2α (10 µM) and IL-1β (10 ng/mL) for 30 min. (C) Analysis of NF-κB protein levels in the cytoplasmic and nuclear fractions. HUtSMCs were pretreated with PDR97 for 30 min, followed by coincubation with PGF2α (10 µM) and IL-1β (10 ng/mL) for 30 min. (D) Quantification of NF-κB protein levels. The relative expression level of NF-κB protein was normalized to the PGF2α- and IL-1β-treated control group. Data are presented as mean ± SEM. Statistical analysis was performed using one-way ANOVA, followed by a nonparametric test. ⁣ ∗ p < 0.05, ⁣ ∗∗ p < 0.01, ⁣ ∗∗∗ p < 0.001, ⁣ ∗∗∗∗ p < 0.0001.

Article Snippet: HUtSMCs were purchased from PromoCell (Cat# C-12575, PromoCell, Heidelberg, Germany).

Techniques: Activation Assay, Luciferase, Activity Assay, Translocation Assay, Expressing, Control