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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 and simultaneous detection of phosphorylated PDGFRβ and ACTB mRNA molecules in PDGF-BB stimulated BJhTert cells.Black circles represent the numbers of RCPs detected per cell (in total ∼85–130 cells per condition), black bars represent 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 ACTB expression, although recorded together.
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pone-0020148-g003: Individual and simultaneous detection of phosphorylated PDGFRβ and ACTB mRNA molecules in PDGF-BB stimulated BJhTert cells.Black circles represent the numbers of RCPs detected per cell (in total ∼85–130 cells per condition), black bars represent 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 ACTB expression, although recorded together.

Mentions: Next, we tested the combined detection of protein modifications and single transcripts (Figure 1). For this purpose phosphorylation of PDGFRβ and ACTB mRNA molecules – an abundantly expressed housekeeping gene to enable a more detailed analysis of detection efficiency – were detected either individually or simultaneously. The cells were fixed and permeabilized before cDNA synthesis and padlock probe hybridization and ligation were performed. After the ACTB cDNA had been targeted, the cells were blocked and stained with in situ PLA reagents required to detect phosphorylated PDGFRβ. Both, reacted padlock probe and in situ PLA probes were amplified in the same reaction by RCA and detected by hybridization of fluorescence labeled detection oligonucleotides. We investigated whether the combined detection shows similar results as when the two detection reactions were run separately, and whether there is a decrease in detection efficiency upon combining the two. BJhTert cells were stimulated with PDGF-BB for 0 min, 5 min or 180 min. The results of the combined detection were consistent with the results of individual staining and the levels of ACTB transcripts were unaffected by stimulation with PDGF-BB, as expected. The combined protocol leads to a decrease in detected signals for both in situ PLA and padlock probes of about 50% compared to when performed in separate reactions (Figure 3).


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 and simultaneous detection of phosphorylated PDGFRβ and ACTB mRNA molecules in PDGF-BB stimulated BJhTert cells.Black circles represent the numbers of RCPs detected per cell (in total ∼85–130 cells per condition), black bars represent 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 ACTB expression, although recorded together.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020148-g003: Individual and simultaneous detection of phosphorylated PDGFRβ and ACTB mRNA molecules in PDGF-BB stimulated BJhTert cells.Black circles represent the numbers of RCPs detected per cell (in total ∼85–130 cells per condition), black bars represent 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 ACTB expression, although recorded together.
Mentions: Next, we tested the combined detection of protein modifications and single transcripts (Figure 1). For this purpose phosphorylation of PDGFRβ and ACTB mRNA molecules – an abundantly expressed housekeeping gene to enable a more detailed analysis of detection efficiency – were detected either individually or simultaneously. The cells were fixed and permeabilized before cDNA synthesis and padlock probe hybridization and ligation were performed. After the ACTB cDNA had been targeted, the cells were blocked and stained with in situ PLA reagents required to detect phosphorylated PDGFRβ. Both, reacted padlock probe and in situ PLA probes were amplified in the same reaction by RCA and detected by hybridization of fluorescence labeled detection oligonucleotides. We investigated whether the combined detection shows similar results as when the two detection reactions were run separately, and whether there is a decrease in detection efficiency upon combining the two. BJhTert cells were stimulated with PDGF-BB for 0 min, 5 min or 180 min. The results of the combined detection were consistent with the results of individual staining and the levels of ACTB transcripts were unaffected by stimulation with PDGF-BB, as expected. The combined protocol leads to a decrease in detected signals for both in situ PLA and padlock probes of about 50% compared to when performed in separate reactions (Figure 3).

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