Protein Quantification Using Resonance Energy Transfer between Donor Nanoparticles and Acceptor Quantum Dots

A homogeneous time-resolved luminescence resonance energy transfer (TR-LRET) assay has been developed to quantify proteins. The competitive assay is based on resonance energy transfer (RET) between two luminescent nanosized particles. Polystyrene nanoparticles loaded with Eu³⁺ chelates (EuNPs) act as donors, while protein-coated quantum dots (QDs), either CdSe/ZnS emitting at 655 nm (QD655-strep) or CdSeTe/ZnS with emission wavelength at 705 nm (QD705-strep), are acceptors. In the absence of analyte protein, in our case bovine serum albumin (BSA), the protein-coated QDs bind nonspecifically to the EuNPs, leading to RET. In the presence of analyte proteins, the binding of the QDs to the EuNPs is prevented and the RET signal decreases. RET from the EuNPs to the QDs was confirmed and characterized with steady-state and time-resolved luminescence spectroscopy. In accordance with the Förster theory, the approximate average donor–acceptor distance is around 15 nm at RET efficiencies, equal to 15% for QD655 and 13% for QD705 acceptor, respectively. The limits of detection are below 10 ng of BSA with less than a 10% average coefficient of variation. The assay sensitivity is improved, when compared to the most sensitive commercial methods. The presented mix-and-measure method has potential to be implemented into routine protein quantification in biological laboratories

Transcriptomic analysis of the UT-SCC-43A, UT-SCC-43B and 43A-SNA oral SCC cell lines.

<p>A) Microarray transcriptomic profiling of UT-SCC-43A, UT-SCC-43B and 43A-SNA cells. Transcripts significantly altered in UT-SCC-43B cells in comparison with UT-SCC-43A cells are indicated by the blue circle and transcripts altered in 43A-SNA cells in comparison with UT-SCC-43A are indicated by the green circle. The number of genes altered in both comparisons is shown in the overlapping area. The number of up-regulated genes is shown on the top and down-regulated genes on the bottom. B) mRNA level alterations for selected epithelial (top) and mesenchymal (bottom) genes. Depicted genes encode following proteins: CDH1 =  E-cadherin, CLDN3 =  Claudin 3, CLDN 7 =  Claudin 7, KRT5 =  Keratin 5, KRT6B  =  Keratin 6b, CDH2 =  N-Cadherin, VIM  =  Vimentin. C) mRNA levels for formins with significant alterations in both UT-SCC-43B and 43A-SNA cells that were subsequently confirmed by RT-PCR. D) mRNA levels for formins confirmed by RT-PCR.</p

FHOD1 siRNA knockdown changes the morphology of UT-SCC-43B cells and reduces the number of stress fibres.

<p>A) Western blotting shows that FHOD1 siRNA treatment significantly reduces FHOD1 expression. The cells are still E-cadherin negative and the expression of N-cadherin is unaltered. B) Cells treated with non-targeting siRNA (upper panel) express FHOD1 and have a mesenchymal phenotype. Stress fibres are abundant. FHOD1 siRNA abolishes FHOD1 staining (lower panel). siRNA treated cells have less actin stress fibres and are morphologically rounder and flatter. Nuclei are stained with DAPI (blue). C) F-actin staining is significantly reduced in FHOD1 siRNA treated cells. Phalloidin staining intensity is reduced by 49%. Bars indicate standard error of mean. *** p<0.0001. AU  =  arbitrary units.</p

FHOD1 is upregulated in clinical oral SCC, as well as <i>in vitro</i> in SCC cells with EMT features.

<p>A) In normal or non-neoplastic stratified squamous epithelium, no FHOD1 can be detected (top). In invasive squamous cell carcinomas, moderate to strong FHOD1 immunoreactivity is seen in spindle-shaped cells at the invasive front that on morphological grounds have undergone EMT (bottom; details in inset). In the tumour bulk, which consists of cells with an epithelial morphology, only weak immunoreactivity is present. Scale bar: 200 μm. B) Western blot analysis of cell lines show that the epithelial SCC cell line UT-SCC-43A does not express detectable FHOD1, while both the spontaneous EMT cell line UT-SCC-43B and the Snail-induced EMT cell line 43A-SNA express FHOD1. UT-SCC-43A expresses the epithelial marker E-cadherin but not N-cadherin, whereas UT-SCC43-B and 43A-SNA express N-cadherin but not E-cadherin. C) F-actin organization of UT-SCC-43A (upper panel) is typically epithelial, with distinct cell submembraneous filaments and scant stress fibres. UT-SCC-43B shows features of mesenchymal organization (middle panel). Cell-cell contacts are few, the cells are elongated and contain lamellopodia, filopodia and stress fibres. The insert (bottom panel) shows that a fraction of FHOD1 co-localizes with stress fibres in UT-SCC-43B cells (arrowheads). Nuclei are stained with DAPI (blue). D) FHOD1 upregulation in UT-SCC-43B cells is dependent of PI3K signalling. Treatment with MEK 1/2 inhibitor U0126 reduces phosphorylation of ERK 1/2 but does not influence FHOD1 expression. In contrast, PI3K inhibition by LY294992 markedly reduces FHOD1 expression. The reduction of p-Akt indicates that the pathway is efficiently inhibited.</p

Expression of FHOD1 in various tissues and cell types.

*<p>0 =  no staining, + =  weak staining, ++ =  moderate staining, +++ =  strong staining.</p

FHOD1 silencing inhibits proteolytic activity and invadopodia formation.

<p>A) Images from a zymography assay performed with untreated, nt siRNA and FHOD1 siRNA treated SCC-43B cells. Degradation of Cy 3 labelled gelatin is reduced in FHOD1 siRNA treated cells. B) Quantification of the degraded area/cell. C) Invadopodia formation is visualized by phalloidin staining, which reveals the comet- or ring-shaped actin structures on the ventral cell surface (arrowheads). Invadopodia are present in a larger proportion of untreated and control cells than in FHOD1 siRNA treated cells. D) Quantification of percentage of cells with invadopodia. (* P<0.05). N.S.  =  not significant.</p