Am9625

cells were washed in ice-cold PBS (Life Technologies, AM9625), 500 µl of cold 80% MeOH (shared by the Metabolite Profiling Core Facility) was added per well of a six-well plate, and the plate was placed at −80 °C for at least 15 min. Following the −80 °C incubation, the plate was scraped on dry ice and the solution was transferred to a 1.5-ml tube and then vortexed for 5 min. To remove cellular debris, the samples were centrifuged at maximum speed for 10 min at 4 °C, and the supernatant was transferred to a new 1.5-ml tube on dry ice. To remove solvents, the samples were lyophilized using Refrigerated CentriVap Benchtop Vacuum Concentrator connected to a CentriVap-105 Cold Trap (Labconco). Metabolite pellets were resuspended in LC-MS grade water (ThermoFisher), and vortexed for 10 min at 4 °C. Samples were centrifuged at 16,000×g for 10 min at 4 °C and supernatant was moved into LC-MS vials. Liquid chromatography and mass spectrometry were performed by the Whitehead metabolomics core.

Prior to mRNA labeling, fixed SW480 cells were blocked with 1% Bovine Serum Albumin (RLBSA50; VWR), 0.1% TWEEN® 20, 1:1,000 Sodium Azide, 0.2 U/ml Protector RNase inhibitor (3335399001; Sigma-Aldrich), and 1 mM DTT in RNAse-free PBS (AM9625; Life Technologies) for 30 min at room temperature. These cells were then washed 3 times with 0.1% TWEEN® 20 in RNAse-free PBS for 5 min each wash at room temperature. Antibody solutions containing 1:1,000 Mouse anti-Tubulin (3873BF; Cell Signaling) and 1:200 Rabbit anti-Vimentin (5741BF; Cell Signaling) in the same blocking buffer were subsequently added to the samples and incubated overnight at 4 °C. Following 3 additional washes with 0.1% TWEEN® 20 in RNAse-free PBS for 5 min each at room temperature, antibody solutions containing fluorescently labeled 1:200 Donkey anti-Mouse Alexa-488 (R37114; Fisher Scientific) and 1:200 Donkey anti-Rabbit TRITC (711-025-152; Jackson Laboratories) in the same blocking buffer were added at room temperature for 1 h. After 3 washes with RNAse-free PBS with 0.1% TWEEN® 20 for 10 min each wash at room temperature, 4% PFA in PBS was added for 15 min at room temperature. These cells were then washed 3 times with 0.1% TWEEN® 20 in PBS at room temperature for 5 min. For mRNA labeling, the previously described methods regarding primary and secondary probe hybridization were utilized.

Eggs, embryos, larvae, juveniles, and adult fish were handled in compliance with applicable national and international standards, according to ZF facility regulation at the University of Bergen. Under normal laboratory conditions, an adult (90+ days post-fertilization dpf) wild-type ZF was held at 28 °C on a 14 h light/10 h dark period. Standard spawning protocol (www.zfin.org, accessed on 18 October 2022) was followed by egg harvesting. Eggs were stored in an E3 medium containing 0.01% methylene blue after harvesting. Embryos and larvae were incubated at 28 °C until 5 dpf. Current regulation does not require permission for testing on ZF embryos before the free-feeding stage (5 dpf). Instead, according to the ZF facility rules, all invasive pain-causing interventions on stages older than 5 dpf was performed under anesthetic conditions. For sample collection, adult ZF were humanely euthanized in 300 mg/L tricaine methanesulfonate MS222 Sigma-Aldrich A-5040 (Steinheim, Germany), and then dissected open. Kidneys were exposed after discarding the viscera under cold 1X PBS Life Technologies AM9625 (Waltham, MA, USA), removed and placed into clean RNAse-free tube prefilled with RNAlater™ Stabilization Solution AM7021(Waltham, MA, USA). Three kidneys were pooled per sample. Samples were kept overnight at 4 °C and stored at −80 °C until further RNA extraction take place.