CDK12 controls transcription at damaged genes and prevents MYC-induced transcription-replication conflicts.

The identification of genes involved in replicative stress is key to understanding cancer evolution and to identify therapeutic targets. Here, we show that CDK12 prevents transcription-replication conflicts (TRCs) and the activation of cytotoxic replicative stress upon deregulation of the MYC oncogene. CDK12 was recruited at damaged genes by PARP-dependent DDR-signaling and elongation-competent RNAPII, to repress transcription.

Development of a live cell assay for the zinc transporter ZnT8.

Zinc is an essential trace element that is involved in many biological processes and in cellular homeostasis. In pancreatic β-cells, zinc is crucial for the synthesis, processing, and secretion of insulin, which plays a key role in glucose homeostasis and which deficiency is the cause of diabetes. The accumulation of zinc in pancreatic cells is regulated by the solute carrier transporter SLC30A8 (or Zinc Transporter 8, ZnT8), which transports zinc from cytoplasm in intracellular vesicles.

Multiplex detection of protein-protein interactions using a next generation luciferase reporter.

Cell-based assays of protein-protein interactions (PPIs) using split reporter proteins can be used to identify PPI agonists and antagonists. Generally, such assays measure one PPI at a time, and thus counterscreens for on-target activity must be run in parallel or at a subsequent stage; this increases both the cost and time during screening. Split luciferase systems offer advantages over those that use split fluorescent proteins (FPs).

Open access to high-content clonogenic analysis.

Image-processing programs are used to identify and classify eukaryotic cell colonies as spots following seeding at low density on dishes or in multiwell plates. The output from such approaches, however, is generally limited to 1-2 parameters, and there is no ability to extract phenotypic information at the single colony level. Furthermore, there is a lack of user-friendly pipelines for analysis of clonogenicity in the context of high-content analysis.

Multiphoton molecular photorelease in click-chemistry-functionalized gold nanoparticles.

Yellow-green controlled photorelease: probes click-linked to peptide-coated gold nanospheres by a triazole ring can be released in living cells under a focused 561 nm laser at low power. Photocleaving follows a three-photon event stimulated by the excitation of the localized surface plasmon resonance.

Single-step bifunctional coating for selectively conjugable nanoparticles.

A single-step method to coat and bifunctionalize water-reduced gold nanoparticles (NPs) with two distinct reactive groups is reported. The coating is based on a peptide that bonds to the NPs surface by its N-cysteine amino acid, terminates with a C-terminal lysine, and stabilizes the colloids, thanks to the surface organization provided by the rest of the non-polar chain. The process yields stable, non-cytotoxic NPs presenting reactive amine and carboxylic groups on the surface; these allow rapid, selective and modular conjugation of virtually any chosen biomolecule or fluorophore.

Simultaneous intracellular chloride and pH measurements using a GFP-based sensor.

Chloride and protons perform important closely related roles in many cellular responses. Here we developed a ratiometric biosensor, ClopHensor, based on a highly chloride-sensitive Aequorea victoria GFP variant that is suited for the combined real-time optical detection of pH changes and chloride fluxes in live cells. We detected high chloride concentration in large dense-core exocytosis granules by targeting ClopHensor to these intracellular compartments.

Quantitative FRET analysis with the EGFP-mCherry fluorescent protein pair.

Fluorescence resonance energy transfer (FRET) between fluorescent proteins (FPs) is a powerful tool to investigate protein-protein interaction and even protein modifications in living cells. Here, we analyze the E(0)GFP-mCherry pair and show that it can yield a reproducible quantitative determination of the energy transfer efficiency both in vivo and in vitro. The photophysics of the two proteins is reported and shows good spectral overlap (Förster radius R(0) = 51 A), low crosstalk between acceptor and donor channels, and independence of the emission spectra from pH and halide ion concentration.

Green fluorescent protein ground states: the influence of a second protonation site near the chromophore.

The photophysical properties of most green fluorescent protein mutants (GFPs) are strongly affected by pH. This effect must be carefully taken into account when using GFPs as fluorescent probes or indicators. Usually, the pH-dependence of GFPs is rationalized on the basis of the ionization equilibrium of the chromophore phenol group.

Spectroscopic and structural study of proton and halide ion cooperative binding to gfp.

This study reports the influence of halogens on fluorescence properties of the Aequorea victoria Green Fluorescent Protein variant S65T/T203Y (E(2)GFP). Halide binding forms a specific nonfluorescent complex generating a substantial drop of the fluorescence via static quenching. Spectroscopic analysis under different solution conditions reveals high halogen affinity, which is strongly dependent on the pH.

Development of a novel GFP-based ratiometric excitation and emission pH indicator for intracellular studies.

We report on the development of the F64L/S65T/T203Y/L231H GFP mutant (E2GFP) as an effective ratiometric pH indicator for intracellular studies. E2GFP shows two distinct spectral forms that are convertible upon pH changes both in excitation and in emission with pK close to 7.0.

Resting smooth muscle cells as a model for studying vascular cell activation.

Vascular smooth muscle (VSM) cells constitute the main structural components of tunica media. Under physiological conditions, these cells display a contractile phenotype and a low proliferative activity. However, they may also acquire a synthetic phenotype and become predominantly proliferative if stimulated under certain stress conditions.

Two dimensional patterning of fluorescent proteins in hydrogels.

This work describes the successful micropatterning of hybrid systems consisting of hydrogel-dispersed optically active and controllable proteins on solid surfaces without degradation of the photophysical properties of the light-emitting biomolecules. It demonstrates the preservation of the luminescence properties of proteins entrapped into isolated microstructures of poly(acrylamide) gel. This way we can exploit both the structural and function-preserving properties of the hydrogels and the functionality of light-emitting proteins.

Identification of active siRNAs against IGF-IR of porcine coronary smooth muscle cells in a heterologous cell line.

Testing of short interfering RNAs (siRNAs) to knock-down gene expression is complicated in primary differentiated cells due to their limited availability and short in vitro life span. The objective of this study was to bypass these limitations by testing selected siRNAs in a heterologous cell line. A plasmid containing a fragment of porcine IGF-I receptor (pIGF-IR) gene cloned downstream of EGFP sequence was constructed and cotransfected with pIGF-IR/siRNAs in HEK 293T human cell line.