human uterine smc contraction assay primary hutsmcs  (PromoCell)

Journal: Science Advances

Article Title: Integrative analysis of noncoding mutations identifies the druggable genome in preterm birth

doi: 10.1126/sciadv.adk1057

Figure Lengend Snippet: ( A ) The algorithm ranked all the 4293 FDR-approved drugs, clinical trial drugs, and preclinical tool compounds based on the predicted effectiveness on treating sPTB. ( B ) The predicted scores and rank percentiles for drugs that had been used or are under clinical trials to treat sPTB. ( C ) The top 50 highly ranked drugs displayed significant functional associations with the preterm birth genes identified in this study, where the bottom ranked 50 drugs exhibited the least functional associations with the preterm birth genes from this study. ( D ) The top 10 candidate drugs predicted by our model for experimental validation. ( E ) The effects on increasing or decrease myometrial cell contractility determined by the collagen gel contraction assay when treating primary human uterine smooth muscle cells (HUtSMCs) with the identified small molecules. The treatment effectiveness was determined in the contractile (stimulated by OXY) or in the quiescence state (PBS). P values were derived from Wilcoxon rank sum tests. ( F ) The dose-response curve for the top candidate drug RKI-1447 in quiescent HUtSMCs. The inset shows the dose-response curve of HUtSMCs treated with different concentrations of RKI-1447. ( G ) Studying the effects of RKI-1447 on the multiscale network identified its functional associations with many muscle proteins with the strongest association with MYLK. ( H ) Docking analysis between RKI-1447 and MYLK confirmed their binding affinity.

Article Snippet: Primary HUtSMCs were purchased from PromoCells (catalog no. C-12575, Thermo Fisher Scientific, MA).

Techniques: Functional Assay, Collagen Gel Contraction Assay, Derivative Assay, Binding Assay

Journal: Redox biology

Article Title: Oxidative stress mediates the inhibitory effects of Manzamine A on uterine leiomyoma cell proliferation and extracellular matrix deposition via SOAT inhibition.

doi: 10.1016/j.redox.2023.102861

Figure Lengend Snippet: Fig. 1. Manz A inhibited uterine leiomyoma in vitro and in vivo. (A) Rat ELT-3 uterine leiomyoma cells and (B) human uterine smooth muscle cells (HUtSMCs) were exposed to the indicated concentrations of Manz A for 24 or 48 h. Cell viability was assessed by an MTS assay. (C) A colony-formation assay was performed to determine the long-term effects of Manz A on the growth of ELT-3 cells. Cells were pretreated with vehicle (0.1% (v/v) DMSO) or Manz A (2.5 or 5 μM) for 48 h and left for seven days to grow in a drug-free medium. Colonies were then stained with Giemsa. (D) The in vivo effect of Manz A was assessed. ELT-3 cells (1.5 × 105) were encapsulated into MTAMs and subcutaneously (s.c.) implanted in female BALB/c mice. Mice were administered with Manzamine A (2 mg/kg/day) or the vehicle control DMSO group (n = 5 for each group) through intraperitoneal (i.p.) injection once daily for ten days. Cell viability (%) was measured by MTT assay and is expressed as the mean ± SD. ***p < 0.001. (E) ELT-3 cells were treated with vehicle (0.1% (v/v) DMSO) or Manz A (2.5 or 5 μM) for 24 h and then subjected to a DNA content analysis by flow cytometry. Representative histograms of the cell cycle distribution are shown in the upper panel. Cell cycle distributions from three independent experiments are quantified in the bottom panel. (F) Protein expressions of cell cycle regulators in response to Manz A treatment in ELT-3 cells were analyzed by western blot. Representative blot images and quantitation of protein levels are shown. Actin was used as an internal control. Data is expressed as the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: The Eker rat-derived uterine leiomyoma cell line Eker Leiomyoma Tumor-3 (ELT-3) [22] (American Type Culture Collection, Manassas, VA, USA) and primary human uterine smooth muscle cells (HUtSMCs) (PromoCell, Heidelberg, Germany) were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% (v/v) fetal bovine serum in a humidified atmosphere containing 95% air and 5% CO2 at 37 ◦C.

Techniques: In Vitro, In Vivo, MTS Assay, Colony Assay, Staining, Control, Injection, MTT Assay, Flow Cytometry, Western Blot, Quantitation Assay