Monitoring On-Target Signaling Responses in Larval Zebrafish - Z-REX Unmasks Precise Mechanisms of Electrophilic Drugs and Metabolites

And so leveraging Z-REX, researchers can interrogate how individual signaling responses are altered in response to a specific reactive ligand engaging with a specific protein of interest in near physiological conditions in Zebrafish larvae. Conventional methods like bulk dose reactive small-molecules causing of targets and toxicity and give no control over ligand target specificity. Z-REX can link a specific ligand target engagement with a specific phenotype with high spatiotemporal precision.

We have recently shown how a reactive metabolite HNE, an associated target and pathway discovered by Z-REX, enables the development of precision isoform-specific kinase inhibitors against triple-negative breast cancers. Begin by aligning the embryos in the grooves of an injection plate filled with fresh 10%Hank's balanced salt solution or HBSS. Submerge the needle tip in the medium, and if required, apply injection pulses to remove air bubbles in the tip For injecting the embryo, penetrate the chorion and yolk sac in a single move and apply an injection pulse.

After injecting the other embryos, rinse them into a new Petri dish containing fresh HBSS media using a squirt bottle. Pool the non-injected embryos into another plate. To perform Z-REX treatment analysis, begin by distributing the injected embryos into 10 centimeter dishes.

In a dark room with red light, replace the medium with 30 milliliters of 10%HBSS with or without one micromolar of the REX probe. Incubate the embryos at 28.5 degrees Celsius in the dark. At 30 hours post fertilization, replace the media in the dark.

After repeating incubation and media replacement twice, expose the embryos to light from a pre-warmed UV lamp swirling every 30 seconds. To perform a phenotypic assay, remove the unfertilized or dead embryos from the plate containing the anesthetized embryos. Using sharp forceps, remove the chorion of the embryos.

Mount the embryos on a 2%agarose plate prepared with 10%HBSS medium and observe under a stereomicroscope. Use ImageJ to count the neutrophil and macrophage number of each fish. Select the Freehand Selection and circle the whole fish, then choose the option Find Maxima to count the fluorescent cells In the present study, Z-REX-treated embryos demonstrated a loss of neutrophils and macrophages.

This effect was absent in the untreated groups. The apoptosis signaling pathway was not triggered in the Halo-P2A-Keap1 group resulting in unaltered macrophage and neutrophil levels. Similar results were also observed in the Halo-TEV-Keap1 mutant that did not respond to the HNE.

Transcriptional and real-time quantitative PCR analyses showed downregulation of immune-related genes and upregulation of antioxidant genes after Z-REX. Immunofluorescence staining showed that the P2A-split-construct had two times more HA tags than the TeV-fusion-construct, indicative of similar expression levels. The expression levels of the wild type and mutant type Halo-TeV-Keap1 were also similar.

In the Z-REX-treated transgenic neutrophil reporter line, colocalization of neutrophils and active caspase 3 was also observed. The most important thing is to have your documents set up correctly. Limiting will really improve your results.

We have adopted Z-REX, an idea to a drug screen platform known as Localis-REX. No other technique so far has been able to achieve such local specific and reactive ligand or target mapping. It has helped us to develop electrophilic drug candidates towards the treatment of breast cancer.

Given the growing importance of localized signaling across many fields, cancer, immunology, aging, et cetera, Z-Rex, the only tool capable of mapping the localized reactive ligand directed events, promises to aid numerous fields.