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AoSMC response to FGF2: Difference between revisions

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|TCSample_description=We provided total RNA (in triplicate) from growth arrested human aortic SMCs (Cell Applications) treated with IL-1beta or FGF-2 for periods of up to 6 hours. SMCs (pool of 3 donors) were grown in 100 mm petri dishes in Waymouth’s medium, pH 7.4, supplemented with 1 mM L-glutamine, 10 units/ml penicillin, 10 mcg/ml streptomycin and 10% fetal bovine serum, at 37°C in a humidified atmosphere of 5% CO2. The cells were rendered growth-quiescent at 80-90% confluency by incubation in serum-free medium for 24h. The SMCs were then exposed to IL-1beta (10ng/ml) or FGF-2 (50ng/ml) for various times up to 6 hours (0, 15, 30, 45, 60, 120, 180, 240, 300 and 360 min). 0 min samples represent growth arrested and unstimulated cells. RNA was harvested using TRIzol reagent, quantitated using a Nanodrop spectrophometer and validated for the transient induction of Egr-1 mRNA (by quantitative real-time PCR) prior to shipment to the Omics Science Center, RIKEN Yokohama Institute (Japan) for CAGE analysis.
|TCSample_description=We provided total RNA (in triplicate) from growth arrested human aortic SMCs (Cell Applications) treated with IL-1beta or FGF-2 for periods of up to 6 hours. SMCs (pool of 3 donors) were grown in 100 mm petri dishes in Waymouth’s medium, pH 7.4, supplemented with 1 mM L-glutamine, 10 units/ml penicillin, 10 mcg/ml streptomycin and 10% fetal bovine serum, at 37°C in a humidified atmosphere of 5% CO2. The cells were rendered growth-quiescent at 80-90% confluency by incubation in serum-free medium for 24h. The SMCs were then exposed to IL-1beta (10ng/ml) or FGF-2 (50ng/ml) for various times up to 6 hours (0, 15, 30, 45, 60, 120, 180, 240, 300 and 360 min). 0 min samples represent growth arrested and unstimulated cells. RNA was harvested using TRIzol reagent, quantitated using a Nanodrop spectrophometer and validated for the transient induction of Egr-1 mRNA (by quantitative real-time PCR) prior to shipment to the Omics Science Center, RIKEN Yokohama Institute (Japan) for CAGE analysis.
|Time_Course=
|Time_Course=
|category_treatment=differentiation
|category_treatment=Differentiation
|collaborators=Levon Khachigian
|collaborators=Levon Khachigian
|description=human_Aortic_smooth_muscle_cell_FGF2
|description=human_Aortic_smooth_muscle_cell_FGF2

Revision as of 12:04, 4 February 2015

Series:IN_VITRO DIFFERENTIATION SERIES
Species:Human (Homo sapiens)
Genomic View:Zenbu
Expression table:FILE
Link to TET:[{{{tet_file}}} TET]
Sample providers :Levon Khachigian
Germ layer:mesoderm
Primary cells or cell line:primary cells
Time span:6 hours
Number of time points:9


Overview

Vascular smooth muscle cells (SMCs) are key components in our blood vessels, and show remarkable plasticity. SMCs are normally growth-quiescent in the normal adult vessels, but are activated by injury, or exposure to growth factors, such as fibroblast growth factor-2 (FGF-2) and pro-inflammatory cytokines, such as interleukin-1beta (IL-1beta). These cues are sensed by these cells through changes in immediate-early gene expression, and can lead to increased proliferation and migration. These responses are associated with the initiation and progression of a range of vascular diseases including atherosclerosis, post-angioplasty restenosis and bypass graft stenosis.

Sample description

We provided total RNA (in triplicate) from growth arrested human aortic SMCs (Cell Applications) treated with IL-1beta or FGF-2 for periods of up to 6 hours. SMCs (pool of 3 donors) were grown in 100 mm petri dishes in Waymouth’s medium, pH 7.4, supplemented with 1 mM L-glutamine, 10 units/ml penicillin, 10 mcg/ml streptomycin and 10% fetal bovine serum, at 37°C in a humidified atmosphere of 5% CO2. The cells were rendered growth-quiescent at 80-90% confluency by incubation in serum-free medium for 24h. The SMCs were then exposed to IL-1beta (10ng/ml) or FGF-2 (50ng/ml) for various times up to 6 hours (0, 15, 30, 45, 60, 120, 180, 240, 300 and 360 min). 0 min samples represent growth arrested and unstimulated cells. RNA was harvested using TRIzol reagent, quantitated using a Nanodrop spectrophometer and validated for the transient induction of Egr-1 mRNA (by quantitative real-time PCR) prior to shipment to the Omics Science Center, RIKEN Yokohama Institute (Japan) for CAGE analysis.

Quality control

Early growth response-1 (Egr-1) is an immediate-early gene (encoding a zinc finger transcription factor) that is poorly expressed in growth-quiescent cells and serves as a marker of cell activation or stress. Total RNA provided to the RIKEN Yokohama Institute was first analysed for Egr-1 expression by qRT-PCR. This demonstrated peak inducible expression after 30 min by FGF-2 and after 60 min by IL-1beta (Fig 1). Transient expression of this biomarker within 30-60 min is supported by the literature (e.g. Zhu et al. 2007).

Figure 1 (qRT-PCR analysis, EGR-1)


CAGE analysis on these samples revealed that Egr-1 underwent transient induction within 30-60 min in response to the growth factor or cytokine (Fig 2). In contrast, CAGE analysis revealed no change in expression of alpha-actin 2 (ACTA2) in response to FGF-2 or IL-1beta within the 6h time frame (Fig 2).


Figure 2 (CAGE analysis, EGR-1 and ACTA2)


CAGE analysis (Fig 3) and subsequent qRT-PCR analysis using separate samples in which Egr-1 was induced (Fig 4) also revealed dynamic changes in the expression of two other prototypic immediate-early genes, c-FOS and FOSB, in response to FGF-2 or IL-1beta (Fig 5).

Figure 3 (CAGE analysis, c-FOS and FOSB)



Figure 4 (qRT-PCR analysis, EGR-1)



Figure 5 (qRT-PCR analysis, c-FOS and FOSB)

Profiled time course samples

Only samples that passed quality controls (Arner et al. 2015) are shown here. The entire set of samples are downloadable from FANTOM5 human / mouse samples


12642-134G5Aortic smooth muscle cell response to FGF200hr00minbiol_rep1
12643-134G6Aortic smooth muscle cell response to FGF200hr15minbiol_rep1
12644-134G7Aortic smooth muscle cell response to FGF200hr30minbiol_rep1
12645-134G8Aortic smooth muscle cell response to FGF200hr45minbiol_rep1
12646-134G9Aortic smooth muscle cell response to FGF201hrbiol_rep1
12647-134H1Aortic smooth muscle cell response to FGF202hrbiol_rep1
12648-134H2Aortic smooth muscle cell response to FGF203hrbiol_rep1
12650-134H4Aortic smooth muscle cell response to FGF205hrbiol_rep1
12651-134H5Aortic smooth muscle cell response to FGF206hrbiol_rep1
12740-135I4Aortic smooth muscle cell response to FGF200hr00minbiol_rep2
12741-135I5Aortic smooth muscle cell response to FGF200hr15minbiol_rep2
12742-135I6Aortic smooth muscle cell response to FGF200hr30minbiol_rep2
12743-135I7Aortic smooth muscle cell response to FGF200hr45minbiol_rep2
12745-135I9Aortic smooth muscle cell response to FGF202hrbiol_rep2
12746-136A1Aortic smooth muscle cell response to FGF203hrbiol_rep2
12748-136A3Aortic smooth muscle cell response to FGF205hrbiol_rep2
12749-136A4Aortic smooth muscle cell response to FGF206hrbiol_rep2
12839-137B4Aortic smooth muscle cell response to FGF200hr15minbiol_rep3
12840-137B5Aortic smooth muscle cell response to FGF200hr30minbiol_rep3
12841-137B6Aortic smooth muscle cell response to FGF200hr45minbiol_rep3
12842-137B7Aortic smooth muscle cell response to FGF201hrbiol_rep3
12843-137B8Aortic smooth muscle cell response to FGF202hrbiol_rep3
12844-137B9Aortic smooth muscle cell response to FGF203hrbiol_rep3
12846-137C2Aortic smooth muscle cell response to FGF205hrbiol_rep3
12847-137C3Aortic smooth muscle cell response to FGF206hrbiol_rep3
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