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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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Simultaneous detection of individual phosphorylated PDGFRβ and DUSP6 mRNA molecules in BJhTert cells.Cells were stimulated for (Ai) 5 min or (Aii) 180 min with PDGF-BB. In situ PLA signals, derived from PDGFRβ phosphorylation, are shown as red dots while DUSP6 molecules are shown as green dots. The nuclei are shown in grey. Inserts represent magnified views over random cells. Scalebars represent 20 µm. (B) Simultaneous detection of individual phosphorylated PDGFRβ and DUSP6 mRNA molecules cells treated with Gleevec or 5-Iodotubercidin at different time points after PDGF-BB stimulation. (Bi) Schematic illustration of the different drug target sites in PDGFRβ pathway and the consequences for signaling and expected RCPs. (Bii) Simultaneous detection of individual phosphorylated PDGFRβ and DUSP6 mRNA molecules in BJhTert cells treated with Gleevec or 5-Iodotubercidin, at different time points after PDGF-BB stimulation. Black circles represent the numbers of RCPs detected per cell (in total ∼100–140 cells per condition), the red 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. The result is presented in separate figures for PDGFRβ phosphorylation and DUSP6 expression, although recorded together.
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pone-0020148-g004: Simultaneous detection of individual phosphorylated PDGFRβ and DUSP6 mRNA molecules in BJhTert cells.Cells were stimulated for (Ai) 5 min or (Aii) 180 min with PDGF-BB. In situ PLA signals, derived from PDGFRβ phosphorylation, are shown as red dots while DUSP6 molecules are shown as green dots. The nuclei are shown in grey. Inserts represent magnified views over random cells. Scalebars represent 20 µm. (B) Simultaneous detection of individual phosphorylated PDGFRβ and DUSP6 mRNA molecules cells treated with Gleevec or 5-Iodotubercidin at different time points after PDGF-BB stimulation. (Bi) Schematic illustration of the different drug target sites in PDGFRβ pathway and the consequences for signaling and expected RCPs. (Bii) Simultaneous detection of individual phosphorylated PDGFRβ and DUSP6 mRNA molecules in BJhTert cells treated with Gleevec or 5-Iodotubercidin, at different time points after PDGF-BB stimulation. Black circles represent the numbers of RCPs detected per cell (in total ∼100–140 cells per condition), the red 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. The result is presented in separate figures for PDGFRβ phosphorylation and DUSP6 expression, although recorded together.

Mentions: Having developed a method that enabled measurement of both protein activity and mRNA expression in single cells, we then tested if the assay would be suitable to detect perturbation of the PDGFRβ-signaling pathway using drugs that affect the pathway at different levels. In that way we were able to investigate if the assay could help in zooming in on the action of a drug. As proof of principle, we examined the effect of Gleevec (a tyrosine kinase inhibitor [13] targeting the PDGFRβ directly) and 5-Iodotubercidin (an inhibitor of ERK2 activation [14], located downstream of PDGFRβ but upstream of the induction of DUSP6 transcription) on phosphorylation of PDGFRβ and DUSP6 expression. As expected, treatment with Gleevec eliminated the increase in signals for both molecules, since it inhibits the activation of the PDGFRβ and thus all downstream events. 5-Iodotubercidin, on the other hand, did only eliminate the upregulation of DUSP6 but not phosphorylation of the PDGFRβ, since it affected the signaling downstream of the receptor but upstream of DUSP6 (Figure 4).


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)

Simultaneous detection of individual phosphorylated PDGFRβ and DUSP6 mRNA molecules in BJhTert cells.Cells were stimulated for (Ai) 5 min or (Aii) 180 min with PDGF-BB. In situ PLA signals, derived from PDGFRβ phosphorylation, are shown as red dots while DUSP6 molecules are shown as green dots. The nuclei are shown in grey. Inserts represent magnified views over random cells. Scalebars represent 20 µm. (B) Simultaneous detection of individual phosphorylated PDGFRβ and DUSP6 mRNA molecules cells treated with Gleevec or 5-Iodotubercidin at different time points after PDGF-BB stimulation. (Bi) Schematic illustration of the different drug target sites in PDGFRβ pathway and the consequences for signaling and expected RCPs. (Bii) Simultaneous detection of individual phosphorylated PDGFRβ and DUSP6 mRNA molecules in BJhTert cells treated with Gleevec or 5-Iodotubercidin, at different time points after PDGF-BB stimulation. Black circles represent the numbers of RCPs detected per cell (in total ∼100–140 cells per condition), the red 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. The result is presented in separate figures for PDGFRβ phosphorylation and DUSP6 expression, although recorded together.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3102075&req=5

pone-0020148-g004: Simultaneous detection of individual phosphorylated PDGFRβ and DUSP6 mRNA molecules in BJhTert cells.Cells were stimulated for (Ai) 5 min or (Aii) 180 min with PDGF-BB. In situ PLA signals, derived from PDGFRβ phosphorylation, are shown as red dots while DUSP6 molecules are shown as green dots. The nuclei are shown in grey. Inserts represent magnified views over random cells. Scalebars represent 20 µm. (B) Simultaneous detection of individual phosphorylated PDGFRβ and DUSP6 mRNA molecules cells treated with Gleevec or 5-Iodotubercidin at different time points after PDGF-BB stimulation. (Bi) Schematic illustration of the different drug target sites in PDGFRβ pathway and the consequences for signaling and expected RCPs. (Bii) Simultaneous detection of individual phosphorylated PDGFRβ and DUSP6 mRNA molecules in BJhTert cells treated with Gleevec or 5-Iodotubercidin, at different time points after PDGF-BB stimulation. Black circles represent the numbers of RCPs detected per cell (in total ∼100–140 cells per condition), the red 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. The result is presented in separate figures for PDGFRβ phosphorylation and DUSP6 expression, although recorded together.
Mentions: Having developed a method that enabled measurement of both protein activity and mRNA expression in single cells, we then tested if the assay would be suitable to detect perturbation of the PDGFRβ-signaling pathway using drugs that affect the pathway at different levels. In that way we were able to investigate if the assay could help in zooming in on the action of a drug. As proof of principle, we examined the effect of Gleevec (a tyrosine kinase inhibitor [13] targeting the PDGFRβ directly) and 5-Iodotubercidin (an inhibitor of ERK2 activation [14], located downstream of PDGFRβ but upstream of the induction of DUSP6 transcription) on phosphorylation of PDGFRβ and DUSP6 expression. As expected, treatment with Gleevec eliminated the increase in signals for both molecules, since it inhibits the activation of the PDGFRβ and thus all downstream events. 5-Iodotubercidin, on the other hand, did only eliminate the upregulation of DUSP6 but not phosphorylation of the PDGFRβ, since it affected the signaling downstream of the receptor but upstream of DUSP6 (Figure 4).

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