Fig. 5. Aromatic residues are required for MYOCD condensate formation and reporter gene expression.

(A) Representative micrographs (max projections) of COS-7 cells expressing MYOCD-mEGFP WT or ΔTAD below or above the critical concentration threshold. Scale bar, 5 μm. (B) Bar chart of the fraction of cells with condensates. Data are mean ± SEM. P values from Mann-Whitney test, ****P ≤ 0.0001. Three biological replicates total, n = 292 (WT) and 106 (ΔTAD). (C) Violin plot of MYOCD concentration and condensate formation in COS-7 cells expressing MYOCD WT or ΔTAD, where each dot represents one nucleus. (D) Representative micrographs (max projections) of COS-7 cells expressing MYOCD-mEGFP WT or ΔTAD above the critical concentration threshold. Scale bar, 5 μm. (E) Bar chart of the fraction of cells with condensates. Data are mean ± SEM. P values from Mann-Whitney test, ****P ≤ 0.0001. n ~ 40. (F) Schematic of MYOCD WT, ΔTAD, aromatic substitution to alanine (FWYtoA) mutants, and TAD domain chimeras with indicated disordered region (FUS and TAF15) with transcription factor (TF)–binding domains (blue box). See fig. S5A for detailed domain architecture of MYOCD. (G and K) Representative micrographs of COS-7 cells expressing indicated MYOCD variants above the critical concentration threshold. (H and L) Bar chart of the fraction of cells with condensates. Data are mean ± SEM. P values represent results of one-way analysis of variance (ANOVA) with Dunn’s multiple comparison test (P values: ns P ≥ 0.05, ****P ≤ 0.0001). (H) n ~ 20. (l) n ~ 15. (I and M) Representative micrographs (max projections) of COS-7 cells expressing the fluorescent reporter and the indicated MYOCD variant above the critical concentration threshold. Scale bar, 5 μm. (J and N) Bar chart of the fraction of cells expressing reporter. Data are mean ± SEM. P values from one-way ANOVA with Dunn’s multiple comparison test (P values: ns P ≥ 0.05, ****P ≤ 0.0001). (I) n ~ 40. (N) n ~ 20.