Hoechst 33528

Prior to fixation cells were washed twice with room temperature 1 × PBS and then fixed in 1 mL of 2% paraformaldehyde (PFA) solution and incubated at 4 °C for 20 min. Following incubation, the cells were washed twice in 1 × PBS. For nuclei staining, 500 μL of freshly prepared Hoechst 33528 (1:500 dilution in PBS; Sigma‐Aldrich) was added to each well and incubated at 37 °C for up to 10 min. Cells were then washed twice with PBS and rinsed once with ddH₂O to remove any salt crystals. Coverslips were mounted face down onto slides containing a drop of Vectashield anti‐fade mounting medium (Vector Laboratories, Peterborough UK).

Throughout cultivation in the CERO 3D Incubator and Bioreactor, representative samples were taken on given days of analysis to determine the growth parameters of spheroids. First, 500 µL of cell suspension were transferred into a 24-well plate and 10 random pictures were taken using a Primovert microscope (Zeiss, Jena, Germany). Spheroid diameter was determined using Fiji [47 (link),48 (link)]. On average, 1295 spheroids (minimum of 601) were analysed per cell type and day of analysis, originating from three independent experiments. In total, 10 µL of suspension were transferred in another well and the number of aggregates in 4 drops per day of analysis was counted manually (spheroid number). After analysis, samples were returned to the culture. Three independent experiments per cell type were conducted. Samples of the spheroids at Days 1, 3, and 7 of culturing were also stained with Hoechst 33528 (Sigma-Aldrich), examined under a Leica DM4000B microscope, and the number of cell nuclei per spheroid area was determined (packing density). On average, 29 spheroids (minimum of 26) were analysed per cell type and day of analysis, originating from two independent experiments.

The primary antibodies used are shown in Table 3. Hoechst 33528 (Sigma #14530) and DAPI (ThermoFisher Scientific, #D1306) were used for nuclear labeling. All secondary antibodies were Pre-adsorbed F(ab)2’ AlexaFluor-conjugated from AbCam.Table 3

Primary antibodies used in this study

Target	Host	Company	Product #	Dilution
Aβ [D54D2]	Rabbit	Cell Signaling Technologies	8243	1:100–1000
Myelin Basic Proteina SMI-99	Mouse	BioLegend	808401	1:50–500
Myelin Basic Proteina SMI-94	Mouse	BioLegend	836502	1:50–500
β-amyloid [6 F/3D]	Mouse	Dako	M087201–2	1:50

^aThese antibodies were used concurrently as per the manufacturer’s recommendation

The dorsal and ventral muscle tissue from 10 animals per species, snap-frozen in liquid nitrogen and stored at -80°C. Subsequently, 100 mg of each sample were pestled and homogenized in 0.01 M potassium phosphate buffer containing 1 mM EDTA in order to analyse the supernatant as previously described in Stange et al. (2020) [43 (link)]; dilution of samples was adapted accordingly. DNA and RNA amount were measured fluorometrically by using either Hoechst 33528 (Sigma-Aldrich) or SYBR Green II RNA Gel Stain (MoBiTec) and quantified against a calf thymus DNA standard or a calf liver RNA standard (Sigma-Aldrich), respectively [44 (link)]. Total protein amount was determined using Pierce^™ BCA Protein Assay Kit (Thermo Fisher Scientific, Inc., USA) according to manufactures instructions. The supernatant of samples was as well used to quantify the activity of specific muscle enzymes. The CK-NAC-Hit kit (IFCC method, BIOMED Labordiagnostik GmbH) was used to measure activity of creatine kinase (CK). The activity of lactate dehydrogenase (LDH) and isocitrate dehydrogenase (ICDH) were assessed by measuring NAD(P)H conversion using a NADPH standard curve. All amounts and activities are given per g muscle.

Colocalization studies were performed according to the detailed methods outlined in Cunningham et al. [47 (link)]. In brief, WT human TASK3 and human TASK3_Tyr205Cys cDNA’s were subcloned into the pAcGFP1-N1 fluorescent vector (Clontech-Takara Bio Europe) to create a fusion construct with GFP and expressed in tsA201 cells using TurboFect transfection reagent (ThermoFisher, Loughborough, UK). Prior to fixing the cells, the plasma membranes of the cells were stained with CellMask Deep Red (CMR) (ThermoFisher, UK) and the nuclei with Hoechst 33528 (Sigma-Aldrich, UK). Coverslips of cells were mounted with Vectashield anti-fade mounting medium (Vector Laboratories, UK).
Confocal microscopy images were taken using a LSM 880 confocal microscope (Carl Zeiss, Oberkochen, Germany) and processed using Zen Black software (Carl Zeiss). Cells were excited with an argon laser at either 561, 488, or 405 nm for the CMR plasma membrane stain, pAcGFP fused channel and Hoechst 33528 stained nuclei, respectively.
Colocalization was determined using Zen Black software and Pearson’s correlation coefficient (PCC) was used to represent the degree of co-localization.

D-Luciferin was from Gold Biotechnology (St. Louis, MO); lentiviral shRNA and puromycin from Santa Cruz (Dallas, TX); 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and Hoechst 33528 from Sigma-Aldrich (St. Louis, MO); mouse gene ST2.0 microarrays and relevant reagents from Affymetrix (Santa Clara, CA); recombinant cytokines and growth factors from Immunotools (Friesoythe, Germany); NF-κB-binding ELISA from Active Motif (La Hulpe, Belgium); bortezomib, IMD-0354, 17-DMAG, and deltarasin from Selleckchem (Houston, TX); G418 from Applichem (Darmstadt, Germany); IL-1β and CXCL1 ELISA from Peprotech (London, UK); and primers from VBC Biotech (Vienna, Austria). Primers, antibodies, and lentiviral shRNA pools are listed in Supplementary Tables 6–8.