ABSTRACT: Analysis by Whole Transcriptome Sequencing of the Effects of LLC1 conditioned medium, LLC1 conditioned plus Calcitriol, non-conditioned medium, and non-conditioned medium plus Calcitriol on mRNA Expression in Primary Human Skeletal Muscle Cells
Project description:Primary human skeletal muscle cells (Lonza) were treated with LLC1 conditioned medium, LLC1 conditioned medium plus Calcitriol, LLC1 non-conditioned medium or LLC1 non-conditioned medium plus Calcitriol for a period of 24 hours prior to isolation of RNA.
Project description:MC3T3 osteoblasts were grown in culture medium in the presence of 10 mM beta-glycerophosphate and and 100 micro g per mL of ascorbic acid and 25% LLC1 conditioned medium or vehicle for 7 days. Cells were lysed an RNA was isolated.
Project description:7- week old male and female, C57BL6 mice received subcutaneous injections of LLC1 lung adenocarcinoma cells or vehicle. Three weeks later, the mice were sacrificed and skeletal muscle was obtained for RNA-seq analysis.
Project description:Mechanistic insights into the molecular events by which exercise enhances the skeletal muscle phenotype are lacking, particularly in the context of type 2 diabetes. Here we unravel a fundamental role for exercise-responsive cytokines (exerkines) on skeletal muscle development and growth in individuals with normal glucose tolerance or type 2 diabetes. Acute exercise triggered an inflammatory response in skeletal muscle, concomitant with an infiltration of immune cells. These exercise effects were potentiated in type 2 diabetes. In response to contraction or hypoxia, cytokines were mainly produced by endothelial cells and macrophages. The chemokine CXCL12 was induced by hypoxia in endothelial cells, as well as by conditioned medium from contracted myotubes in macrophages. We found that CXCL12 was associated with skeletal muscle remodeling after exercise and differentiation of cultured muscle. Collectively, acute aerobic exercise mounts a non-canonical inflammatory response, with an atypical production of exerkines, which is potentiated in type 2 diabetes.
Project description:The interplay between skeletal muscle and bone is primarily mechanical, however, biochemical crosstalk by secreted mediators has recently gained increased attention. The aim of this study was to investigate metabolic effects of conditioned medium from osteoblasts (OB-CM) on myotubes and vice versa. Human skeletal muscle cells incubated with OB-CM showed increased glucose uptake and oxidation, and expression of the glucose transporter (GLUT) 1, while fatty acid uptake and oxidation, and expression of the fatty acid transporter CD36 were decreased. This was supported by proteomic analysis where expression of proteins involved in glucose uptake, glycolytic pathways and TCA cycle were enhanced, and expression of several proteins involved in fatty acid metabolism were reduced. Similar effects on energy metabolism were observed in human bone marrow stromal cells differentiated to osteoblastic cells incubated with conditioned medium from myotubes (SKM-CM), with increased glucose uptake and reduced oleic acid uptake. Proteomic analyses of the two conditioned media revealed many common proteins. Thus, our data may indicate a shift in fuel preference from fatty acid to glucose metabolism in both cell types, induced by conditioned media from the opposite cell type, possibly indicating a more general pattern in communication between these tissues.
Project description:To study the effect of myoblast-derived paracrine factors on cardiomyocyte gene expression, microarray analysis was performed from isolated rat fetal cardiomyocyte cultures. These cultures were treated for 24 hours with fresh culture medium (control), skeletal myoblast-conditioned medium, or cardiac fibroblast-conditioned medium.
Project description:We treated the prostate tumor cell line LNCaP with serum from prostate cancer patients that participated in a 12-week physical exercise and nutrional study. Exercise was conducted in form of whole-body electromyostimulation (WB-EMS). All patients from the control and the intervention group received nutritional counseling to achieve high-protein dietary intake (please refer to Schink, Herrmann et al, 2018; PMID: 30208857). Serum from all patients was taken at study entry and at the end of the intervention in week 12. Additionally, human primary skeletal muscle cells were treated with electric pulse stimulation to simulate in vitro exercise. EPS-conditioned medium was then also used to incubate prostate tumor cells. Treatment with either study patient serum or conditioned myotube medium was done for 24 h and for 72 h. Total RNA was isolated and cancer-related gene expression was analyzed using Nanostring technology.
Project description:Mechanically dissociated IPF lung explant derived cellular suspensions were cultured in 50% senescent lung fibroblast conditioned medium + 50% fibroblast complete medium (DMEM + 15% FBS + antiboitics) and 10 µM of Y27632. After approximately 2 weeks, cells were passaged and cultured overnight in Pneumacult Ex Plus medium (STEMCELL technologies).
Project description:Primary cilia (PC) are important signaling hubs and their deregulation is associated with various diseases. The 2P-CF cultures plus starvation mimic the in vivo proportion of PC on colonic fibroblasts (CF) in an intact colon and thus allow to explore the biology of PC. We generated conditioned starvation medium from colons of DSS treated (termed inflammatory conditioned medium, i.e. inf-CM) and untreated control animals (termed co-CM) and cultured 2P-CF in those media for 24 hours. 30-40% of 2P-CF starved in control medium displayed PC, whereas PC number on 2P-CF starved in inf-CM was consistently lower. We performed RNAseq analysis of 2P-CF starved in inf-CM or in co-CM to gain insight in the molecular traits of those cell cultures.
Project description:The Skeletal muscle is a metabolic active tissue that secretes various proteins. These so called myokines act auto-, para- and endocrine affecting muscle physiology and exert systemic effects on other tissues and organs. Myokines are also described to play a crucial role in the pathophysiology of metabolic diseases. Combining three different mass spectrometry based non-targeted and one antibody based targeted approach we investigated the secretome of differentiated primary human skeletal muscle cells derived from adult donors. A total of 548 non-redundant proteins were detected by combined proteomic profiling. Expression was confirmed on mRNA level for 501. Stringent consecutive filtering recruiting several database, i.e. SignalP, SecretomeP and ER_retention signals, the computational analysis assigned 306 as secretory proteins including 33 potentially novel myokines. This comprehensive profiling study of the human skeletal muscle secretome expands our knowledge of the composition of the human myokinome and further highlights the pivotal role of myokines in the regulation of multiple biological processes. We performed gene expression microarray analysis of primary human myotubes derived from twelve healthy individuals
