title: Simultaneous visualization of both signaling cascade activity and end-point gene expression in single cells.
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.
authors.affiliation: Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden.
authors.forename: Irene
authors.initials: I
authors.lastname: Weibrecht
authors.forename: Ida
authors.initials: I
authors.lastname: Grundberg
authors.forename: Mats
authors.initials: M
authors.lastname: Nilsson
authors.forename: Ola
authors.initials: O
authors.lastname: Söderberg
mesh_terms: D001549:Benzamides; D002460:Cell Line; D054642:Dual Specificity Phosphatase 6 / Q000235:genetics; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D000068877:Imatinib Mesylate; D007700:Kinetics; D008024:Ligands; D057054:Molecular Imaging / Q000379:methods*; D010766:Phosphorylation / Q000187:drug effects; D010879:Piperazines / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020797:Receptor, Platelet-Derived Growth Factor beta / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D059010:Single-Cell Analysis / Q000379:methods*; D013997:Time Factors
chemical_list: D001549:Benzamides; D008024:Ligands; D010879:Piperazines; D011743:Pyrimidines; D012333:RNA, Messenger; D000068877:Imatinib Mesylate; D020797:Receptor, Platelet-Derived Growth Factor beta; C517035:DUSP6 protein, human; D054642:Dual Specificity Phosphatase 6