Eclipse ti2

F-actin and G-actin measurement was performed as described.⁶⁵ D28 hCOs were dissociated into single cells using the same method used for scRNA-seq. Harvested cells were resuspended in organoid maturation medium and passed through a 40 μm filter strainer to obtain single cells. Cells then seeded at a density of 0.05 million/well on 24-well size coverslips coated with hESC qualified Matrigel. After two days in culture, cells were fixed with 4% PFA for 15 min. Permeabilization and blocking of the samples are performed as mentioned in the section on immunofluorescence microscopy. After blocking, cells were stained with antibody Alexa Fluor™ 647 Phalloidin (F-actin, ThermoFisher Scientific, Cat#A22287), Deoxyribonuclease I, Alexa Fluor™ 488 Conjugate (G-actin, ThermoFisher Scientific, Cat# D12371) and DAPI (ThermoFisher Scientific, Cat#D1306) for 2 h at room temperature. Cells were then washed thrice in 1×PBS and mounted with FluorSave^TM (Merck, Cat#345789) for imaging (Nikon, Eclipse Ti2; Zeiss, Axio Observer Z1 and Nikon, Eclipse TE2000-E). The same exposure settings were used to acquire images from WT and FEZ1-null groups. Images were analyzed with ImageJ/Fiji with quantification details in the following section. At least three independent experiments were performed.

H9c2 cells and adult rat cardiomyocytes, which were isolated as described previously
²⁷ (link) were seeded on glass coverslips. Staining using different dyes including Cyto‐ID, (1:1000, Enzo Life Sciences, #51031), Magic Red (1:260, ImmunoChemistry Technologies, #942), DalGreen (1:1000, Dojindo, #D675) DapRed (1:1000, Dojindo, #D677), cleaved caspase 3/7 (1:500, ThermoFisher, #C10423), and ReadyProbe (ThermoFisher, #R37609) was performed according to manufacturer's protocols and DAPI (HCS NuclearMask Blue Stain, ThermoFisher, #H10325) was added simultaneously. Cells were fixed in 10% formalin (Sigma, #HT501128) after treatment and mounted with ProLong Gold Antifade Mountant (ThermoFisher, #P36930). Slides were observed under Nikon Eclipse Ti2, Zeiss LSM700 confocal microscope at 60×, Evos FL Auto 2 and for continuous measurements Cellnsight CX7 Platform (ThermoFisher, #CX7A1110) was used. Co‐localization analysis was performed with an ImageJ JACOP plug‐in. At least five random fields from one coverslip/well, counting at least 30 cells were average in each independent experiment. The bar chart represents mean ± SEM values from at least three independent experiments.