Measurement of the specific lipid content of attached cells in microtiter cultures

Laughton, Craig W.

doi:10.1016/0003-2697(86)90258-7

Cited by 30 publications

(14 citation statements)

References 15 publications

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“…The lipid(s) extracted with isopropyl alcohol from the stained cells by the procedure described below was subjected to thin-layer chromatography. Only traces of non-TG lipids were evident in the isopropanol extracts, results that are in agreement with those of Laughton (18). The author recommended validation of the specificity with each cell type used.…”

Section: Methodssupporting

confidence: 78%

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Melatonin inhibits fatty acid-induced triglyceride accumulation in ROS17/2.8 cells: implications for osteoblast differentiation and osteoporosis

Sánchéz-Hidalgo¹,

Lu²,

Tan³

et al. 2007

American Journal of Physiology-Regulatory, Integrative and Comp

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Melatonin is produced not only by the pineal gland but by cells of the bone marrow. Moreover, melatonin is known to promote osteogenic differentiation in several cell line models and in multipotential bone marrow mesenchymal stem cells. Fatty acids have been independently shown to direct such cells to acquire the phenotype and molecular characteristics of adipocytes. To examine the effect of melatonin on intracellular triglyceride accumulation, an indicator of adipogenic differentiation in the rat osteoblast-like ROS17/2.8 cell line, cells were incubated with added oleic acid (100 muM), fixed and stained with Oil Red O. Cellular lipid accumulation was quantitated by an Oil Red O method highly specific for triglycerides and expressed as a triglyceride accumulation index (TGAI, triglyceride per cell). Melatonin in nanomolar concentrations inhibited oleic acid-induced triglyceride accumulation. To identify the mechanism by which melatonin reduces triglyceride accumulation, cells were incubated with the two melatonin receptor antagonists, luzindole and S20928, or forskolin, a stimulator of adenylyl cyclase and cAMP production. These compounds prevented the inhibitory effect of melatonin on triglyceride accumulation, indicating that melatonin acts through known melatonin receptor-mediated mechanisms. In view of the previously demonstrated positive effects of melatonin in promoting osteoblastic differentiation in ROS17/2.8 cells and their reciprocal adipocytic differentiation induced by fatty acids, our observations may serve to relate the known age-related decreases of melatonin production, the shift in the bone marrow toward an adipocytic line of cell development, and the development of osteoporosis during aging.

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Section: Methodssupporting

confidence: 78%

“…The ORO staining method as described previously (16,18,30) or adapted by us with identified minor modification, is highly specific for TG (18,30). Similarly, the only significant lipid in the extracted ORO-stained lipid droplets was TG.…”

Section: Methodsmentioning

confidence: 99%

Melatonin inhibits fatty acid-induced triglyceride accumulation in ROS17/2.8 cells: implications for osteoblast differentiation and osteoporosis

Sánchéz-Hidalgo¹,

Lu²,

Tan³

et al. 2007

American Journal of Physiology-Regulatory, Integrative and Comp

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“…Glycoprotein synthesis was determined by alcian blue staining (44). For adipogenesis, the accumulation of lipid droplets, a hallmark of functional adipogenesis, was detected by oil red staining (44) and quantified at 14 days of culture, as described (45). Briefly, the stain solution was removed, and cells were rinsed in 500 l of 60% isopropyl alcohol for 5 s. To extract dye, 700 l of 60% isopropyl alcohol was added per well, and sealed plates were shaken for 2 h. The extracted dye was quantitated spectrophotometrically at 510 nm.…”

Section: Methodsmentioning

confidence: 99%

Fibroblast Growth Factor Receptor 2 Promotes Osteogenic Differentiation in Mesenchymal Cells via ERK1/2 and Protein Kinase C Signaling

Miraoui

Oudina

Petite

et al. 2009

Journal of Biological Chemistry

135

134

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Mesenchymal stem cells (MSCs)3 have the potential to differentiate into different lineages, including osteoblasts, chondroblasts, and adipocytes (1-7). The osteoblast differentiation program of MSCs is characterized by cell recruitment, which is followed by timely expressed genes including Runx2, alkaline phosphatase (ALP), type I collagen (ColA1), and osteocalcin (OC), which is associated with extracellular matrix mineralization (8 -10). The program of MSC osteogenic differentiation can be induced by soluble molecules such as bone morphogenetic proteins (BMPs) or Wnt proteins that activate several signaling pathways to trigger osteoblast differentiation (11-15). Although various downstream signals are known to promote osteogenic differentiation (16 -20), the molecular mechanisms that control the early stages of MSC osteoblast differentiation are not fully elucidated.Fibroblast growth factors (FGFs) play an important major role in the control of cell proliferation, differentiation, and survival in several tissues including bone (21-24). Notably, FGF2 was found to promote cell growth and osteoblast differentiation in bone marrow-derived mesenchymal cells (25,26). Consistent with an important role of FGF signaling in the control of osteoprogenitor cells, deletion of FGF2 in mice results in decreased bone marrow stromal cell osteogenic differentiation and altered bone formation (27). The actions of FGFs are highly dependent on high affinity FGF receptors (FGFRs) (28). FGF binding to FGFRs leads to receptor dimerization and phosphorylation of intrinsic tyrosine residues, which leads to activation of several signal transduction pathways including phospholipase C␥ (PLC␥), mitogen-activated protein kinases (MAPK), and phosphatidylinositol 3-kinase (PI3K) (29,30). In bone, activation of extracellular-related kinase (ERK1/2) MAPK and protein kinase C (PKC) was found to enhance osteoblast gene expression (31, 32). The important role of FGFR signaling in bone formation is highlighted by the finding that gain-of-function mutations in FGFRs results in premature cranial osteogenesis (33, 34). FGFR1 was recently shown to be an important transducer of FGF signals in proliferating osteoblasts (35). In contrast, activated FGFR2 was shown to enhance osteoblast differentiation in Apert syndromic craniosynostosis (36 -41). However, the role of FGFR2 signaling in osteogenic differentiation of mesenchymal stem cells is yet to be elucidated.In the present study, we investigated the specific role of FGFR2 signaling on osteoblast commitment and differentiation

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“…The effect of antisense RNAs on the morphological differentiation of 3T3-L1 cells was assessed by culturing the transformants in differentiation-inducing medium and then staining these cells with the lipid-specific dye oil red 0 (24 control cells and cells expressing clone 5 antisense RNA ranged from 13-to 18-fold in dexamethasone-treated cells and from 14-to 28-fold in untreated TA1 cells.…”

Section: Resultsmentioning

confidence: 99%

Reduced expression of AP27 protein, the product of a growth factor-repressible gene, is associated with diminished adipocyte differentiation.

Wenz¹,

Hinck²,

Cannon³

et al. 1992

Proc. Natl. Acad. Sci. U.S.A.

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We have recently characterized an adipocyte cDNA (clone 5) that is enhanced in expression by environmental and hormonal conditions favoring adipogenic differentiation. Moreover, certain agents including fibroblast growth factor and phorbol 12-myristate 13-acetate (but not epidermal growth factor) markedly inhibit clone 5 gene expression and prevent TA1 cell differentiation. These results led us to propose that a threshold level of the clone 5 gene product (AP27 protein) is required for triggering adipocyte differentiation. We have constructed vectors that direct the synthesis ofclone S antisense RNA to reduce the levels of AP27 in adipogenic cell lines TA1 and 3T3-L1. We show here that when these cells express clone S antisense RNA, they fail to undergo morphological differentiation, whereas adipogenesis is unaffected in cells expressing antisense fi-actin or ferritin heavy-chain RNA. We further show that cells expressing clone S antisense RNA (but not the other antisense RNAs) are unable to induce the expression of characteristic "adipocyte-specific" mRNAs. The level of inhibition of differentiation by clone 5 antisense RNA correlates with decreased levels of AP27 protein. These results provide strong evidence that expression of AP27 is linked to adipogenic differentiation and that AP27 may be a component of an as-yet-uncharacterized signal-transduction pathway required for the triggering of adipocyte differentiation.Adipogenic stem cells, such as 3T3-L1 (1) and TA1 (2), are widely used to study the mechanisms by which hormonal and environmental signals control the switch from a progenitor cell to its fully differentiated counterpart. Such cell lines resemble fibroblasts while growing at low density, but upon reaching confluence the cells stop growing and in time take on the morphological and biochemical characteristics of mature adipocytes (3). Although recent advances have produced insights into the transcriptional mechanisms mediating differentiation-specific gene expression (4,5), there is still a paucity of data relating to the mechanisms by which hormonal and environmental signals are transduced to the elements mediating adipocyte-specific gene expression.Many groups have presented studies in which growth factors were shown to inhibit the differentiation of determined (especially myogenic) stem cell lines (6-8). We have recently reported that the differentiation of TA1 cells is markedly suppressed by fibroblast growth factor (FGF) and the phorbol ester 12-myristate 13-acetate (PMA), in a growthindependent fashion, as reflected by the inability of the cells to undergo morphological differentiation or to accumulate adipocyte-specific RNAs. We also described the characteristics of a specific mRNA, clone 5, the expression of which is suppressed by these same agents in undifferentiated, preconfluent cultures. This property, in addition to its induction by differentiation-promoting factors such as glucocorticoids and high cell density, suggested to us that the putative protein product of this mRNA (refe...

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Measurement of the specific lipid content of attached cells in microtiter cultures

Cited by 30 publications

References 15 publications

Melatonin inhibits fatty acid-induced triglyceride accumulation in ROS17/2.8 cells: implications for osteoblast differentiation and osteoporosis

Melatonin inhibits fatty acid-induced triglyceride accumulation in ROS17/2.8 cells: implications for osteoblast differentiation and osteoporosis

Fibroblast Growth Factor Receptor 2 Promotes Osteogenic Differentiation in Mesenchymal Cells via ERK1/2 and Protein Kinase C Signaling

Reduced expression of AP27 protein, the product of a growth factor-repressible gene, is associated with diminished adipocyte differentiation.

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