Fig 2: Intracellular RBM20 localization in RBM20 Mutant iPSC-CMs.a Immunofluorescence for RBM20 in WT and RBM20 mutant iPSC-CMs. Spinning disk confocal, scale bar: 5 µm. Representative micrographs from n > 3 experiments. b Structured illumination microscopy of DNA (magenta) and RBM20 (green). Left WT image represents X–Y maximum intensity projection, right images represent X–Z 3D projections. Yellow arrowheads indicate perinuclear localization of RBM20 in mutant cells. Yellow stars indicate the nuclear localization of RBM20. Scale bar: 5 µm. Representative micrographs from n > 3 experiments. c 3D localization analysis of RBM20 from 3D-SIM images. Magenta bars (left bar above genotype) indicate percent nuclear localization, green bars (right bar above genotype) indicate percent cytoplasmic localization. Data represents per-cell averages from N = 2 independent experiments with n = 4 cells per genotype. Data represented as mean values ± SEM.

Fig 3: Model of mutant RBM20 differential splicing and P-body impacts in dilated cardiomyopathy.Proposed model for the impact of wild-type and mutant RBM20 on nuclear regulation of splicing, based on RNA-Seq and eCLIP data, as compared to the cytoplasmic role of mutant RBM20 on P-body formation and 3'UTR association with mRNAs implicated in granule formation.

Fig 4: Generation and functional characterization of mutant iPSC-CMs.a Targeted genomic region of RBM20. The location of R636 and the 8 nucleotides deleted in the 8-bp Del HMZ line are highlighted. b Genomic sequences of the generated RBM20 HTZ and HMZ iPSC lines. c Genomic sequence of the generated RBM20 8-bp Del HMZ line (KO). d iPSC-CM electrophysiological parameters from multi-electrode array (MEA) recordings. Color-coded paired replicates represent biologically independent experiments run in parallel. N = 3 biologically independent experiments. Data represented as mean values ± SEM. e Representative 3D-engineered heart tissues (3D-EHT) from WT and RBM20 mutant cell lines. Phase-contrast microscopy, scale bar: 1 mm. The location of flexible and glass-stiffened silicone posts is indicated. f Contractile metrics of 3D-EHTs. WT: n = 4, HTZ: n = 6, HMZ: n = 13, KO: n = 10. Data represented as mean values ± SEM. Statistical significance was calculated using one-way ANOVA with Dunnett’s multiple comparisons test.

Fig 5: Intracellular localization of RBM20 mutant iPSC-CMs and association with P-bodies.a Localization of RBM20 (green) and DDX6 (processing bodies, magenta). High-magnification insets highlight co-localization. Spinning disk confocal, scale bar: 5 µm. Representative micrographs from n > 3 independent biological experiments. b DDX6/RBM20 co-localization is quantified via Pearson's coefficient of co-localization. Data from N = 3 biological independent experiments. Statistical significance was calculated using one-way ANOVA with Dunnett’s multiple comparisons test. Data represented as mean values ± SEM. c Structured Illumination microscopy of DDX6 (left), RBM20 (middle), and merged channels (DDX6: magenta, RBM20: green). White arrows indicate co-localization; yellow asterisk indicates RBM20 not associated with processing bodies. Scale bar: 0.5 µm. Representative micrographs from n = 2 independent biological experiments. d Assessment of stress granules via G3BP1 localization. iPSC-CMs were analyzed in basal conditions (e.g., normal media) and in response to stress (1 mM sodium Arsenate treatment for 1 h). Spinning disk confocal, scale bar: 5 µm. Representative micrographs from n > 3 independent biological experiments. e Structured illumination microscopy analysis of RBM20 (yellow) and G3BP1 stress granules (cyan) in sodium-arsenate-treated iPSC-CMs. Scale bar: 5 µm. Representative micrographs from n = 2 independent biological experiments. Source data are provided as a Source Data file.