Bdm 2 3 butanedione monoxime

Degradation groups were created as described above. All images were taken using 96-h-old animals. Adult animals were anesthetized by being placed in 10 µl of 7.5 mM Levamisole (Sigma-Aldrich) and 0.225 M BDM (2,3-butanedione monoxime) (Sigma-Aldrich) on glass microscope slide containing a 2% agar pad. After animals were paralyzed, a 1.5 coverslip was placed on top the agar pad. A Leica SP8 white light laser confocal microscope and 63× oil immersion lens was used for imaging. Step size was 0.3 µm. GFP was excited using a 488 nm wavelength laser with emitted light collected through a 490-778 nm bandpass filter. mNeonGreen was excited using a 506 nm wavelength laser with emitted light collected through a 512-742 nm bandpass filter. For each condition, four to five animals were scored for the presence of GFP or mNeonGreen. Final figures were generated using ImageJ (National Institutes of Health, Bethesda, MD, USA).

C. elegans wild-type (N2) animals obtained from the Caenorhabditis Genetics Center were used for this study. The animals were maintained using standard normal growth conditions and procedures^{29 (link)}. For experimental purpose L4 animals were grown at 21 °C until reaching adult stage. To visualize the worm outer surface the young adults were first stained with the fluorescent lipophilic dye DiI (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate) according to reference³⁰ . For coating the worm surface with beads, the DiI pre-stained worms were incubated for 1 hr in amine-modified fluorescent beads (Invitrogen F8764, with a diameter of 0.2 µm) diluted 1:500 in M9 buffer. Prior to the experiment the worms were immobilized by 40 min treatment with 15 mg/ml BDM (2,3-butanedione monoxime, Sigma-Aldrich). Using cover glass with a 2% agarose pad, the paralyzed worms were then glued on to the edge of a second cover slip using dermabond glue (2-octyl cyanoacrylate, Suturonline.com) and subsequently immersed in M9 buffer.

Images of fluorescently tagged fusion proteins were captured in live C. elegans using a Zeiss LSM800 confocal microscope (Carl Zeiss, Germany). Worms were immobilized on 2% agarose pad using a mixture of 7.5 mM levamisole (Sigma-Aldrich) and 0.225M BDM (2,3-butanedione monoxime) (Sigma-Aldrich). Images were analyzed with Zen software (Carl Zeiss) or Image J (NIH, USA). Definition of each parameter is as follows (Mizumoto and Shen, 2013a (link)): DA8/9 overlap: a distance between the most anterior DA9 synapse and the most posterior DA8 synapse, DA8 asynaptic domain: a distance from commissure to the most posterior DA8 synapse, DA9 synaptic domain: a distance between the most anterior and posterior DA9 synapses. Middle L4 (judged by the stereotyped shape of developing vulva) animals were used for quantification. Averages were taken from at least 20 samples. For GFP::Utrophin-CH, we measured the length from the posterior end of dorsal axon to the anterior end of GFP::Utrophin-CH domain. For each marker strain, the same imaging setting (laser power, gain pinhole) and image processing were used for comparing different genotypes.

Around 10 mM hydroxyurea (Sigma-Aldrich, #H8627) was dissolved in FASW; the solution was renewed twice a day. Muscle contraction was inhibited as follows: with 1 or 5 µM blebbistatin (Sigma-Aldrich, #B0560) diluted from a 34 mM stock solution in DMSO, or with 8 mM BDM (2,3-Butanedione monoxime; Sigma-Aldrich, #B0753) in FASW, or with 400 µM menthol (Sigma-Aldrich, #M2772) diluted from a 1M stock solution in ethanol. The β-catenin/Tcf interaction inhibitor PKF118-310 (Lepourcelet et al., 2004 (link); Sigma Aldrich, #K4394) was used at a final concentration of 0.7–0.8 µM, diluted from a 15 mM stock solution in DMSO.

After pollen germination for 3 h, LDs in pollen tubes were stained with neutral lipid stain Nile Red (0.3 µg mL⁻¹) for 15 min before microscopic observation. For pharmacological treatments, pollen germinated for 3 h was submerged in liquid pollen germination medium supplemented with 50 mM 2,3-butanedione monoxime (BDM) (Sigma, Livonia, MI, USA), 10 µM oryzalin (Merck, Kenilworth, NJ, USA), 50 µM brefeldin A (Sigma, Livonia, MI, USA), 40 µM cytochalasin D (Sigma, Livonia, MI, USA), 100 µM SMIFH2 (Sigma, Livonia, MI, USA), and 3 nM latrunculin B (Calbiochem, San Diego, CA, USA) for 15 min. The drugs and the Nile Red stain were washed away before confocal observation.

Porcine hearts were explanted at the end of final surgery after euthanization by i.v. injection of potassium chloride in deep anesthesia. Tissue samples of the anterior right atrium were immediately transferred into preoxygenated Ca²⁺-free Tyrode’s solution [100 mM NaCl, 10 mM KCl, 1.2 mM KH₂PO₄, 5 mM MgSO₄, 50 mM taurine, 5 mM 3-(N-morpholino) propanesulfonic acid (MOPS) and 20 mM glucose, pH 7.0 with NaOH] supplemented with 2,3-butanedione monoxime (BDM, 30 mM; Sigma-Aldrich, St. Louis, MO, United States). Isolation of single atrial cardiomyocytes was performed as previously described (Schmidt et al., 2015 (link)). Atrial tissue samples were rinsed three times for 3 min in Ca²⁺-free Tyrode’s solution. The solutions were preoxygenated with 100% O₂ at 37°C. Subsequently aliquots were digested with collagenase type I (288 U/ml; Worthington) and protease type XXIV (5 mg/ml; Sigma Aldrich) for 15 min before Ca²⁺ concentration was increased to 0.2 mM and samples were stirred for another 35 min in protease-free solution. Finally, single rod-shaped cardiomyocytes could be obtained and resuspended in storage medium [20 mM KCl, 10 mM KH₂PO₄, 10 mM glucose, 70 mM K glutamate, 10 mM β-hydroxybutyrate, 10 mM taurine, 10 mM ethylene glycol tetraacetic acid (EGTA), and 1% albumin] prior to electrophysiological characterization in patch clamp experiments.