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AoSMC response to IL1b: 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=activation
|category_treatment=Activation
|collaborators=Levon Khachigian
|collaborators=Levon Khachigian
|description=human_Aortic_smooth_muscle_cell_IL1b
|description=human_Aortic_smooth_muscle_cell_IL1b
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|time_points=0hr
|time_points=0hr
|time_span=6 hours
|time_span=6 hours
|timepoint_design=early focus
|timepoint_design=Early focus
|tissue_cell_type=Aortic smooth muscle
|tissue_cell_type=Aortic smooth muscle
|zenbu_config=http://fantom.gsc.riken.jp/zenbu/gLyphs/#config=Nrtf4wSeLpqlaXpQB5sxjD
|zenbu_config=http://fantom.gsc.riken.jp/zenbu/gLyphs/#config=Nrtf4wSeLpqlaXpQB5sxjD
}}
}}

Revision as of 12:05, 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:10


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


12652-134H6Aortic smooth muscle cell response to IL1b00hr00minbiol_rep1 (LK31)
12653-134H7Aortic smooth muscle cell response to IL1b00hr15minbiol_rep1 (LK34)
12654-134H8Aortic smooth muscle cell response to IL1b00hr30minbiol_rep1 (LK37)
12655-134H9Aortic smooth muscle cell response to IL1b00hr45minbiol_rep1 (LK40)
12656-134I1Aortic smooth muscle cell response to IL1b01hrbiol_rep1 (LK43)
12658-134I3Aortic smooth muscle cell response to IL1b03hrbiol_rep1 (LK49)
12659-134I4Aortic smooth muscle cell response to IL1b04hrbiol_rep1 (LK52)
12660-134I5Aortic smooth muscle cell response to IL1b05hrbiol_rep1 (LK55)
12661-134I6Aortic smooth muscle cell response to IL1b06hrbiol_rep1 (LK58)
12750-136A5Aortic smooth muscle cell response to IL1b00hr00minbiol_rep2 (LK32)
12751-136A6Aortic smooth muscle cell response to IL1b00hr15minbiol_rep2 (LK35)
12752-136A7Aortic smooth muscle cell response to IL1b00hr30minbiol_rep2 (LK38)
12753-136A8Aortic smooth muscle cell response to IL1b00hr45minbiol_rep2 (LK41)
12754-136A9Aortic smooth muscle cell response to IL1b01hrbiol_rep2 (LK44)
12755-136B1Aortic smooth muscle cell response to IL1b02hrbiol_rep2 (LK47)
12756-136B2Aortic smooth muscle cell response to IL1b03hrbiol_rep2 (LK50)
12757-136B3Aortic smooth muscle cell response to IL1b04hrbiol_rep2 (LK53)
12758-136B4Aortic smooth muscle cell response to IL1b05hrbiol_rep2 (LK56)
12759-136B5Aortic smooth muscle cell response to IL1b06hrbiol_rep2 (LK59)
12848-137C4Aortic smooth muscle cell response to IL1b00hr00minbiol_rep3 (LK33)
12849-137C5Aortic smooth muscle cell response to IL1b00hr15minbiol_rep3 (LK36)
12850-137C6Aortic smooth muscle cell response to IL1b00hr30minbiol_rep3 (LK39)
12851-137C7Aortic smooth muscle cell response to IL1b00hr45minbiol_rep3 (LK42)
12853-137C9Aortic smooth muscle cell response to IL1b02hrbiol_rep3 (LK48)
12855-137D2Aortic smooth muscle cell response to IL1b04hrbiol_rep3 (LK54)
12857-137D4Aortic smooth muscle cell response to IL1b06hrbiol_rep3 (LK60)
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