A Zebrafish Chemical Suppressor Screening Identifies Small Molecule Inhibitors of the Wnt/β-catenin Pathway

SummaryGenetic screening for suppressor mutants has been successfully used to identify important signaling regulators. Using an analogy to genetic suppressor screening, we developed a chemical suppressor screening method to identify inhibitors of the Wnt/β-catenin signaling pathway. We used zebrafish embryos in which chemically induced β-catenin accumulation led to an “eyeless” phenotype and conducted a pilot screening for compounds that restored eye development. This approach allowed us to identify geranylgeranyltransferase inhibitor 286 (GGTI-286), a geranylgeranyltransferase (GGTase) inhibitor. Our follow-up studies showed that GGTI-286 reduces nuclear localization of β-catenin and transcription dependent on β-catenin/T cell factor in mammalian cells. In addition to pharmacological inhibition, GGTase gene knockdown also attenuates the nuclear function of β-catenin. Overall, we validate our chemical suppressor screening as a method for identifying Wnt/β-catenin pathway inhibitors and implicate GGTase as a potential therapeutic target for Wnt-activated cancers

Deformation mechanism of high-density polyethylene probed by in situ Raman spectroscopy

The microscopic mechanism of high-density polyethylene under uniaxial drawing is investigated using in situ Raman spectroscopy. From the peak shifts of the symmetric and anti-symmetric C-C stretching modes, it is found that the load sharing on the polymer chain in the yielding region is anisotropic with stretching along the chain and compression perpendicular to the chain. The orientation functions (〈P2〉 and 〈P4〉) as well as the orientation distribution function (N(θ)) are determined from the polarized Raman spectra. The molecular orientation with cold drawing is found to proceed more effectively for lower crystallinity specimens. In the yielding region, it is also found that N(θ) has a maximum at the polar angle θ = 30-70°. This peculiar behavior in the microscopic scale is explained by the preferential collapse of spherulites and the existence of lamellar clusters as the bulky mobile units

Platelet Supernatant Suppresses LPS-Induced Nitric Oxide Production from Macrophages Accompanied by Inhibition of NF-κB Signaling and Increased Arginase-1 Expression

<div><p>We previously reported that mouse bone marrow-derived macrophages (BMDMs) that had been co-cultured with platelets exhibited lower susceptibility to bacterial lipopolysaccharide (LPS) and produced lower levels of nitric oxide (NO) and inflammatory cytokines including TNF-α and IL-6. The suppression of macrophage responses was mediated, at least in part, by platelet supernatant. In the present study, we assessed phenotypic changes of BMDMs induced by incubation with the supernatant from thrombin-activated platelets (PLT-sup) and found that BMDMs cultured with PLT-sup (PLT-BMDMs) expressed a lower level of inducible NO synthase (iNOS) and a higher level of arginase-1, both of which are involved in the L-arginine metabolism, upon stimulation with LPS or zymosan. We also examined possible modulation of the NF-κB signaling pathway and observed suppression of IκBα phosphorylation and a decrease of NF-κB p65 expression in LPS-stimulated PLT-BMDMs. These results suggest that PLT-sup suppresses inflammatory responses of BMDMs via negative regulation of NF-κB signaling leading to lowered expression of iNOS and enhanced L-arginine catabolism by arginase-1.</p></div

Expression of iNOS and arginase-1 in BMDMs after LPS stimulation.

<p>(A) PLT-BMDMs and control BMDMs (2.5 × 10⁶ cells) were stimulated with LPS (50 ng/mL) for 24 h, and the gene expressions of <i>Nos2</i> and <i>Arg1</i> were analyzed by RT-qPCR with the relative standard curve method using <i>Gapdh</i> as an internal control. The gene expression is represented as the value relative to gene expression in the original BMDMs. Experiments were performed in quintuplicate and repeated four times. The data are presented as the mean ± SEM. ***p < 0.005 vs. control BMDMs. Representative results of the four experiments are shown. (B) PLT-BMDMs and control BMDMs (2.5 × 10⁶ cells) were stimulated with LPS (50 ng/mL) for 0–24 h. Cells were then lysed in 1 × SDS sample buffer, and the cell lysates were subjected to western blotting analysis with antibodies against iNOS, arginase-1 or GAPDH. The relative intensity of each iNOS or arginase-1 band after normalization to the levels for GAPDH is shown in the lower panel. Experiments were repeated four times, and representative results are shown. (C) PLT-BMDMs and control BMDMs (2.5 × 10⁶ cells) were stimulated with zymosan (25 or 100 μg/mL) for 12 h, and then cell lysates were subjected to western blotting analysis with antibodies against iNOS, arginase-1 or GAPDH. <i>C</i>, control BMDMs; <i>P</i>, PLT-BMDMs. The relative intensity of each iNOS or arginase-1 band after normalization to the levels for GAPDH is shown in the lower panel. Experiments were repeated four times, and representative results are shown.</p

Attenuation of LPS-induced NO production from BMDMs by PLT-sup.

<p>BMDMs (2.5 × 10⁶ cells) were cultured for 24 h with PLT-sup (PLT-BMDMs) or 0.5 U/mL thrombin alone (control BMDMs) in a 3.5 cm dish, and stimulated with complete medium containing LPS (50 ng/mL) for 0–24 h. The production levels of NO₂^- in the culture supernatant were determined by Griess reaction using a calibration curve for NaNO₂. Experiments were performed in quintuplicate and repeated four times. Data are presented as the mean ± SEM. *p < 0.05, **p < 0.01 vs. control BMDMs. Representative results of the four experiments are shown.</p