Figure 1.
(A) Upon stimulation with PDGF-BB the PDGFRβ becomes autophosphorylated at various sites. This promotes downstream signaling to ERK activation. Phosphorylated ERK translocates into the nucleus and enhances DUSP6 expression. Upon fixation of the cells DUSP6 mRNA is reverse transcribed utilizing an LNA-modified primer. Subsequently the cDNA is made accessible through RNase H digestion. The single standed cDNA stays attached to the mRNA-primer by the 5′-end of the primer that contains the LNA bases, which is not recognized by RNase H – to allow hybridization of padlock probes. (B) After ligation of the padlock probe to a circular DNA molecule, primary antibodies directed against the PDGFRβ and phosphorylated tyrosines, followed by addition of secondary PLA probes, are applied for detection of the phosphorylated PDGFRβ. If the antibodies and probes are bound in close proximity two circularization oligonucleotides can hybridize to the PLA probes. (C) This molecule can subsequently also be joined by ligation, giving rise to another circular DNA molecule. (D) Both DNA circles are then simultaneously amplified by RCA, primed from either the cDNA or the oligonucleotide attached to the PLA probe. The resulting bundles of DNA (amplified ∼1000 times) are still attached to the place where the RCA was primed and can be detected by hybridization of fluorescence labeled oligonucleotides (different fluorophores (green and pink stars) for the two types of circles), resulting in bright spots easily distinguishable from background.
Figure 2.
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.
Figure 3.
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.
Figure 4.
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.