scholarly journals A simple trypsin resistance assay for muscle and other cell fusion.

1986 ◽  
Vol 34 (7) ◽  
pp. 959-962 ◽  
Author(s):  
G Denning ◽  
A B Fulton
Keyword(s):  
Gene Expression ◽  
Cell Fusion ◽  
Muscle Cells ◽  
Cell Loss ◽  
Lugol’S Iodine ◽  
Edta Solution ◽  
Specific Stage ◽  
Independent Assay ◽  
Study Development

Muscle cells fusing in vitro have long provided biologists with a tool to study development and gene expression. However, many such studies used morphological assays of cell fusion. We present here a method for assaying fusion at a specific, operationally defined step. Muscle cells grown in monolayer are exposed to trypsin-EDTA solution at 37 degrees C; the trypsin is inactivated, the cells fixed in Lugol's iodine, and 200 to 300 nuclei are counted as being single or multiple. The presence of EDTA is important under standard conditions for muscle culture; however, little difference is seen in divalent cation-depleted cultures. Therefore, for consistency EDTA can be included in all assays. Samples are stable for over 24 hr, with no cell loss from trypsinization or fixation. This assay exploits a specific stage of muscle fusion, trypsin-resistant contact, to provide a rapid, simple, and observer-independent assay for an early state of muscle fusion. The assay can be used to measure fusion between any nucleated cells.

scholarly journals OncogenicGata1causes stage-specific megakaryocyte differentiation delay

2019 ◽  
Author(s):  
Gaëtan Juban ◽  
Nathalie Sakakini ◽  
Hedia Chagraoui ◽  
Qian Cheng ◽  
Kelly Soady ◽  
...  
Keyword(s):  
Gene Expression ◽  
Es Cells ◽  
Erythroid Differentiation ◽  
Detailed Examination ◽  
S Phase ◽  
Myeloproliferative Disorder ◽  
Specific Gene ◽  
Specific Stage

AbstractThe megakaryocyte/erythroid Transient Myeloproliferative Disorder (TMD) in newborns with Down Syndrome (DS) occurs when N-terminal truncating mutations of the hemopoietic transcription factor GATA1, that produce GATA1short protein (GATA1s), are acquired early in development. Prior work has shown that murine GATA1s, by itself, causes a transient yolk sac myeloproliferative disorder. However, it is unclear where in the hemopoietic cellular hierarchy GATA1s exerts its effects to produce this myeloproliferative state. Here, through a detailed examination of hemopoiesis from murine GATA1s ES cells and GATA1s embryos we define defects in erythroid and megakaryocytic differentiation that occur relatively in hemopoiesis. GATA1s causes an arrest late in erythroid differentiationin vivo, and even more profoundly in ES-cell derived cultures, with a marked reduction of Ter-119 cells and reduced erythroid gene expression. In megakaryopoiesis, GATA1s causes a differentiation delay at a specific stage, with accumulation of immature, kit-expressing CD41himegakaryocytic cells. In this specific megakaryocytic compartment, there are increased numbers of GATA1s cells in S-phase of cell cycle and reduced number of apoptotic cells compared to GATA1 cells in the same cell compartment. There is also a delay in maturation of these immature GATA1s megakaryocytic lineage cells compared to GATA1 cells at the same stage of differentiation. Finally, even when GATA1s megakaryocytic cells mature, they mature aberrantly with altered megakaryocyte-specific gene expression and activity of the mature megakaryocyte enzyme, acetylcholinesterase. These studies pinpoint the hemopoietic compartment where GATA1s megakaryocyte myeloproliferation occurs, defining where molecular studies should now be focussed to understand the oncogenic action of GATA1s.Scientific CategoryHaematopoiesis and Stem CellsKey PointsGATA1s-induced stage-specific differentiation delay increases immature megakaryocytesin vivoandin vitro, during development.Differentiation delay is associated with increased numbers of cells in S-phase and reduced apoptosis.

scholarly journals SM22α Promoter Targets Gene Expression to Vascular Smooth Muscle Cells In Vitro and In Vivo

2000 ◽  
Vol 6 (11) ◽  
pp. 983-991 ◽  
Author(s):  
Levent M. Akyürek ◽  
Zhi-Yong Yang ◽  
Kazunori Aoki ◽  
Hong San ◽  
Gary J. Nabel ◽  
...  
Keyword(s):  
Gene Expression ◽  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Muscle Cells

scholarly journals Sox6 as a new modulator of renin expression in the kidney

2020 ◽  
Vol 318 (2) ◽  
pp. F285-F297 ◽  
Author(s):  
Mohammad Saleem ◽  
Conrad P. Hodgkinson ◽  
Liang Xiao ◽  
Juan A. Gimenez-Bastida ◽  
Megan L. Rasmussen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Stromal Cells ◽  
Expression Profiling ◽  
Muscle Cells ◽  
Afferent Arteriole ◽  
Adult Kidney

Juxtaglomerular (JG) cells, major sources of renin, differentiate from metanephric mesenchymal cells that give rise to JG cells or a subset of smooth muscle cells of the renal afferent arteriole. During periods of dehydration and salt deprivation, renal mesenchymal stromal cells (MSCs) differentiate from JG cells. JG cells undergo expansion and smooth muscle cells redifferentiate to express renin along the afferent arteriole. Gene expression profiling comparing resident renal MSCs with JG cells indicates that the transcription factor Sox6 is highly expressed in JG cells in the adult kidney. In vitro, loss of Sox6 expression reduces differentiation of renal MSCs to renin-producing cells. In vivo, Sox6 expression is upregulated after a low-Na+ diet and furosemide. Importantly, knockout of Sox6 in Ren1d+ cells halts the increase in renin-expressing cells normally seen during a low-Na+ diet and furosemide as well as the typical increase in renin. Furthermore, Sox6 ablation in renin-expressing cells halts the recruitment of smooth muscle cells along the afferent arteriole, which normally express renin under these conditions. These results support a previously undefined role for Sox6 in renin expression.

243. In vitro culture significantly reduces differences in gene expression profiles between myometrial and fibroid smooth muscle cells

2005 ◽  
Vol 17 (9) ◽  
pp. 96
Author(s):  
M. Zaitseva ◽  
P. A. W. Rogers
Keyword(s):  
Gene Expression ◽  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Cultured Cells ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Muscle Cells ◽  
Compare Gene Expression

Fibroids are benign neoplasms of the smooth muscle cells of the uterus. Cultured myometrial (M) and fibroid (F) smooth muscle cells (SMC) have been widely used as a model for the study of fibroid growth. Although it has been shown that FSMC can behave differently in culture to MSMC, it is not clear how relevant the cultured cells and their responses are to the in-vivo situation. The aim of the present study was to compare gene expression profiles of M and F tissue to cells isolated from the same tissue and cultured for up to 3 passages. M and F were collected from hysterectomy specimens (n = 6), part was snap frozen for RNA and the rest used to isolate SMC, which were cultured for 3 passages and RNA was collected at passage 0 (P0) and 3 (P3). 36 microarrays were performed on 8K human cDNA slides, 6 per each specimen (3 for M and 3 for F: tissue, cell at P0 and P3) against reference RNA. Analysis revealed significant differences between tissues and cultured cells. Independent clustering assigned tissues versus cells into two distinct groups based on their expression profiles. Parametric ANOVA with Benjamini-Hochberg correction and post-hoc testing was used to determine similarities and differences between tissues and cells. 128 genes were found to be statistically different between M and F tissue, 66 between MSMC and FSMC at P0, and only 9 at P3. More than 1100 genes were significantly changed between tissues and cultured cells, with 648 genes common between both M and F cells at P0 and P3. Similar numbers of genes were up regulated as were down regulated. Expression profiles of genes of interest including estrogen receptor α and progesterone receptor were also validated using real-time PCR. This is the first study to compare gene expression of in vivo and in vitro fibroid and myometrial SMC. The results demonstrate that large changes occur in SMC gene expression in culture, reducing differences between myometrial and fibroid cells. This study indicates that results of in vitro studies should be interpreted with caution as many genes have an altered gene expression profile in culture.

Effect of c-myc gene expression on early inducible reactions required for erythroid differentiation in vitro

1988 ◽  
Vol 8 (12) ◽  
pp. 5545-5548
Author(s):  
T Kaneko-Ishino ◽  
T U Kume ◽  
H Sasaki ◽  
M Obinata ◽  
M Oishi
Keyword(s):  
Gene Expression ◽  
Dimethyl Sulfoxide ◽  
Cell Fusion ◽  
Erythroid Differentiation ◽  
Metallothionein Promoter ◽  
Myc Gene ◽  
Suppressive Action ◽  
Mouse Erythroleukemia

By employing cell fusion between two genetically marked mouse erythroleukemia (MEL) cells in which an artificially introduced c-myc gene had been placed under the control of human metallothionein promoter, we investigated the mechanism of the suppressive action of c-myc gene expression in erythroid differentiation. The results indicated that the expression of the c-myc gene blocked the induction of dimethyl sulfoxide-inducible activity, one of the two early activities required for triggering the differentiation.

scholarly journals Posttranslational control of membrane-skeleton (ankyrin and alpha beta-spectrin) assembly in early myogenesis.

1985 ◽  
Vol 100 (5) ◽  
pp. 1726-1735 ◽  
Author(s):  
W J Nelson ◽  
E Lazarides
Keyword(s):  
Cell Fusion ◽  
Early Time ◽  
Muscle Cells ◽  
Membrane Skeleton ◽  
Intermediate Filament Protein ◽  
Adult Chicken ◽  
Anion Transporter ◽  
Alpha Spectrin ◽  
Alpha Beta

Adult chicken skeletal muscle cells express polypeptides that are antigenically related to alpha-spectrin (Mr 240,000) and beta-spectrin (Mr 220,000-225,000), the major components of the erythrocyte membrane-skeleton, and to ankyrin (Mr 237,000; also termed goblin in chicken erythrocytes), which binds spectrin to the transmembrane anion transporter in erythrocytes. Comparative immunoblotting of SDS-solubilized extracts of presumptive myoblasts and fully differentiated myotubes cultured in vitro demonstrated that there is a dramatic accumulation of ankyrin and alpha- and beta-spectrin during myogenesis and a concomitant switch in the subunit composition of spectrin from alpha gamma to alpha beta. Analysis of early time points in myogenesis (12-96 h) revealed that these changes occur shortly after the main burst of cell fusion. To determine the temporal relationship between cell fusion and the accumulation of ankyrin and alpha- and beta-spectrin, we treated presumptive myoblasts with 2 mM EGTA, which resulted in the complete inhibition of cell fusion. The incorporation of [35S]methionine into total protein and, specifically, into alpha-, gamma-, and beta-spectrin remained the same in EGTA-treated and control cells. Analysis by immunoblotting of the amounts of ankyrin and alpha- and beta-spectrin in fusion-blocked cells revealed that there was no effect on accumulation for the first 19 h. However, there was then a dramatic cessation in their accumulation, and thereafter, the amount of each protein at steady state remained constant. Upon release from the EGTA block, the cells fused rapidly (less than 11 h), and the accumulation of ankyrin and alpha- and beta-spectrin was reinitiated after a lag period of 3-5 h at a rate similar to that in control cells. The inhibition in the accumulation of newly synthesized ankyrin, alpha-spectrin, and beta-spectrin in EGTA-treated myoblasts was not characteristic of all structural proteins, since the accumulation of the muscle-specific intermediate filament protein desmin was the same in control and fusion-blocked cells. These results show that in myogenesis, the synthesis of ankyrin and alpha- and beta-spectrin and their accumulation as a complex, although concurrent, are not coupled events. We hypothesize that the extent of assembly of these components of the membrane-skeleton in muscle cells is determined by a control mechanism(s) operative at the posttranslational level that is triggered near the time of cell fusion and the onset of terminal differentiation.

scholarly journals Effect of c-myc gene expression on early inducible reactions required for erythroid differentiation in vitro.

1988 ◽  
Vol 8 (12) ◽  
pp. 5545-5548 ◽  
Author(s):  
T Kaneko-Ishino ◽  
T U Kume ◽  
H Sasaki ◽  
M Obinata ◽  
M Oishi
Keyword(s):  
Gene Expression ◽  
Dimethyl Sulfoxide ◽  
Cell Fusion ◽  
Erythroid Differentiation ◽  
Metallothionein Promoter ◽  
Myc Gene ◽  
Suppressive Action ◽  
Mouse Erythroleukemia

By employing cell fusion between two genetically marked mouse erythroleukemia (MEL) cells in which an artificially introduced c-myc gene had been placed under the control of human metallothionein promoter, we investigated the mechanism of the suppressive action of c-myc gene expression in erythroid differentiation. The results indicated that the expression of the c-myc gene blocked the induction of dimethyl sulfoxide-inducible activity, one of the two early activities required for triggering the differentiation.

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