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Simultaneous visualization of both signaling cascade activity and end-point gene expression in single cells.

Weibrecht I, Grundberg I, Nilsson M, Söderberg O - PLoS ONE (2011)

Bottom Line: As proof-of-principle, we demonstrated the utility of the method for simultaneous detection of phosphorylated PDGFRβ and DUSP6/MKP-3 mRNA molecules in individual human fibroblasts upon PDGF-BB stimulation.Further we applied drugs disrupting the PDGFRβ signaling pathway at various sites to show that this combined method can concurrently monitor the molecular effect of the drugs, i.e. inhibition of downstream signaling from the targeted node in the signaling pathway.Due to its ability to detect different types of molecules in single cells in situ the method presented here can contribute to a deeper understanding of cell-to-cell variations and can be applied to e.g. pinpoint effector sites of drugs in a signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden.

ABSTRACT
We have developed an approach for simultaneous detection of individual endogenous protein modifications and mRNA molecules in single cells in situ. For this purpose we combined two methods previously developed in our lab: in situ proximity ligation assay for the detection of individual protein interactions and -modifications and in situ detection of single mRNA molecules using padlock probes. As proof-of-principle, we demonstrated the utility of the method for simultaneous detection of phosphorylated PDGFRβ and DUSP6/MKP-3 mRNA molecules in individual human fibroblasts upon PDGF-BB stimulation. Further we applied drugs disrupting the PDGFRβ signaling pathway at various sites to show that this combined method can concurrently monitor the molecular effect of the drugs, i.e. inhibition of downstream signaling from the targeted node in the signaling pathway. Due to its ability to detect different types of molecules in single cells in situ the method presented here can contribute to a deeper understanding of cell-to-cell variations and can be applied to e.g. pinpoint effector sites of drugs in a signaling pathway.

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Individual detection of single phosphorylated PDGFRβ and DUSP6 molecules in BJhTert cells.(A) Detection of individual phosphorylated PDGFRβ using in situ PLA in PDGF-BB-stimulated cells. Black circles represent the numbers of RCPs detected in individual cells (in total ∼90–130 cells per condition), the black bar represents the median of the population and grey boxes the 25% and 75% quartiles. The experiment is a representative example of three replicate experiments. (B) Detection of individual DUSP6 mRNA molecules using padlock probes in BJhTert cells stimulated with PDGF-BB for different length of time. Black circles represent the numbers of RCPs detected in individual cells (in total ∼70–180 cells per condition), the black bar represents the median of the population and grey boxes the 25% and 75% quartiles. The experiment is a representative example of three replicate experiments.
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pone-0020148-g002: Individual detection of single phosphorylated PDGFRβ and DUSP6 molecules in BJhTert cells.(A) Detection of individual phosphorylated PDGFRβ using in situ PLA in PDGF-BB-stimulated cells. Black circles represent the numbers of RCPs detected in individual cells (in total ∼90–130 cells per condition), the black bar represents the median of the population and grey boxes the 25% and 75% quartiles. The experiment is a representative example of three replicate experiments. (B) Detection of individual DUSP6 mRNA molecules using padlock probes in BJhTert cells stimulated with PDGF-BB for different length of time. Black circles represent the numbers of RCPs detected in individual cells (in total ∼70–180 cells per condition), the black bar represents the median of the population and grey boxes the 25% and 75% quartiles. The experiment is a representative example of three replicate experiments.

Mentions: To determine the kinetics of PDGFRβ phosphorylation and DUSP6 expression upon stimulation with PDGF-BB, BJhTert cells were starved for 48 h prior to treatment with 100 ng ml−1 PDGF-BB. The cells were stimulated for different length of time (0 min–240 min) and subsequently fixed. Thereafter the phosphorylation of PDGFRβ was investigated using in situ PLA with one antibody directed against the receptor and one against phosphorylated tyrosine residues. In parallel, the expression on DUSP6 transcripts was monitored by padlock probes on separate slides (Figure 2). As expected, the phosphorylation of PDGFRβ first increased substantially during the initial time points, peaking at 5–10 min after stimulation, before it gradually decreased again towards background levels due to internalization of the receptor [9]. In accordance with what has been reported before [10], DUSP6 transcript levels remained constant for the first 30 min of stimulation, and then showed an increase until 3 h. Interestingly, the distribution in numbers of detected molecules per cell in the cell population is quite large, especially for PDGFRβ phosphorylation in response to stimulation. Regarding DUSP6 expression, not all cells are responding to stimulation with PDGF-BB to the same extent. It rather appears that, for the time investigated, some cells burst with transcription while in others the signal is not propagated to the induction of DUSP6 expression.


Simultaneous visualization of both signaling cascade activity and end-point gene expression in single cells.

Weibrecht I, Grundberg I, Nilsson M, Söderberg O - PLoS ONE (2011)

Individual detection of single phosphorylated PDGFRβ and DUSP6 molecules in BJhTert cells.(A) Detection of individual phosphorylated PDGFRβ using in situ PLA in PDGF-BB-stimulated cells. Black circles represent the numbers of RCPs detected in individual cells (in total ∼90–130 cells per condition), the black bar represents the median of the population and grey boxes the 25% and 75% quartiles. The experiment is a representative example of three replicate experiments. (B) Detection of individual DUSP6 mRNA molecules using padlock probes in BJhTert cells stimulated with PDGF-BB for different length of time. Black circles represent the numbers of RCPs detected in individual cells (in total ∼70–180 cells per condition), the black bar represents the median of the population and grey boxes the 25% and 75% quartiles. The experiment is a representative example of three replicate experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3102075&req=5

pone-0020148-g002: Individual detection of single phosphorylated PDGFRβ and DUSP6 molecules in BJhTert cells.(A) Detection of individual phosphorylated PDGFRβ using in situ PLA in PDGF-BB-stimulated cells. Black circles represent the numbers of RCPs detected in individual cells (in total ∼90–130 cells per condition), the black bar represents the median of the population and grey boxes the 25% and 75% quartiles. The experiment is a representative example of three replicate experiments. (B) Detection of individual DUSP6 mRNA molecules using padlock probes in BJhTert cells stimulated with PDGF-BB for different length of time. Black circles represent the numbers of RCPs detected in individual cells (in total ∼70–180 cells per condition), the black bar represents the median of the population and grey boxes the 25% and 75% quartiles. The experiment is a representative example of three replicate experiments.
Mentions: To determine the kinetics of PDGFRβ phosphorylation and DUSP6 expression upon stimulation with PDGF-BB, BJhTert cells were starved for 48 h prior to treatment with 100 ng ml−1 PDGF-BB. The cells were stimulated for different length of time (0 min–240 min) and subsequently fixed. Thereafter the phosphorylation of PDGFRβ was investigated using in situ PLA with one antibody directed against the receptor and one against phosphorylated tyrosine residues. In parallel, the expression on DUSP6 transcripts was monitored by padlock probes on separate slides (Figure 2). As expected, the phosphorylation of PDGFRβ first increased substantially during the initial time points, peaking at 5–10 min after stimulation, before it gradually decreased again towards background levels due to internalization of the receptor [9]. In accordance with what has been reported before [10], DUSP6 transcript levels remained constant for the first 30 min of stimulation, and then showed an increase until 3 h. Interestingly, the distribution in numbers of detected molecules per cell in the cell population is quite large, especially for PDGFRβ phosphorylation in response to stimulation. Regarding DUSP6 expression, not all cells are responding to stimulation with PDGF-BB to the same extent. It rather appears that, for the time investigated, some cells burst with transcription while in others the signal is not propagated to the induction of DUSP6 expression.

Bottom Line: As proof-of-principle, we demonstrated the utility of the method for simultaneous detection of phosphorylated PDGFRβ and DUSP6/MKP-3 mRNA molecules in individual human fibroblasts upon PDGF-BB stimulation.Further we applied drugs disrupting the PDGFRβ signaling pathway at various sites to show that this combined method can concurrently monitor the molecular effect of the drugs, i.e. inhibition of downstream signaling from the targeted node in the signaling pathway.Due to its ability to detect different types of molecules in single cells in situ the method presented here can contribute to a deeper understanding of cell-to-cell variations and can be applied to e.g. pinpoint effector sites of drugs in a signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden.

ABSTRACT
We have developed an approach for simultaneous detection of individual endogenous protein modifications and mRNA molecules in single cells in situ. For this purpose we combined two methods previously developed in our lab: in situ proximity ligation assay for the detection of individual protein interactions and -modifications and in situ detection of single mRNA molecules using padlock probes. As proof-of-principle, we demonstrated the utility of the method for simultaneous detection of phosphorylated PDGFRβ and DUSP6/MKP-3 mRNA molecules in individual human fibroblasts upon PDGF-BB stimulation. Further we applied drugs disrupting the PDGFRβ signaling pathway at various sites to show that this combined method can concurrently monitor the molecular effect of the drugs, i.e. inhibition of downstream signaling from the targeted node in the signaling pathway. Due to its ability to detect different types of molecules in single cells in situ the method presented here can contribute to a deeper understanding of cell-to-cell variations and can be applied to e.g. pinpoint effector sites of drugs in a signaling pathway.

Show MeSH