M33089

10,000 HEK293 FRT were seeded in 96-well plates (M33089; Thermo Fisher Scientific) and induced with tetracycline for 24 h. The next day, cells were transfected with LZTR1 siRNA or control siRNA. 72 h later, we removed the medium. Hoechst 33342 (H3570; Thermo Fisher Scientific) was diluted in PBS in 1:2,000 dilution and added to the cells. The cells were then incubated for 10 min in the incubator. Cells were imaged using the Opera Phenix microscope. Images were further analysed in the Columbus Image Data Storage and Analysis System. The media cell fluorescent per mean fluorescent per well was then determined for each condition. Finally, the siRNA control was used to normalise the data. The experiment was performed in biological triplicate, and the graph was generated using Prism software (GraphPad Prism 9.2.0).

We used a previously described protocol (Matsumoto et al., 2019 (link)) for immunostaining and clearing of the organoids. Briefly, the organoids were fixed overnight in 4% paraformaldehyde followed by delipidation by successive overnight incubations in CUBIC-L solution (50%, 100%) at 37°C. The organoids were then blocked in 3% bovine serum albumin and incubated for 2 d in rabbit anti-Nkx2.5 primary antibody diluted 1:200 in blocking solution. Excess primary antibody was removed using PBST (0.2% Triton X-100 diluted in 1× PBS). The organoids were then incubated again for 2 d in secondary antibody (1:200, Alexa 647 goat-anti-rabbit; Invitrogen, A21244) and DAPI (1 µg/mL; Sigma, P9542) diluted in blocking solution. Samples were cleared for imaging in 96-well optical plates (Thermo Fisher, M33089) using CUBIC-R solution as described previously (Matsumoto et al., 2019 (link)). Confocal images were obtained on a Zeiss LSM 880 inverted confocal microscope with AiryScan (Jena, Germany).