Fig. 6: Decreased association of CBs with HLBs in EAF1-mutant cells.

a, b EAF1 C-terminal region and MED26 N-terminal region are required for CB–HLB association. Wild-type cells and EAF1-mutant cells (a) or MED26-mutant cells (b) were fixed with paraformaldehyde, and immunofluorescence staining for Coilin and NPAT was performed. Scale bar, 5 μm. Enlarged images for representative particles are shown in the lower part of each image. Evaluation of CB–HLB association in each mutant cell line is shown in the right panels. The NPAT particles were extracted from images. The area of NPAT particles occupied by Coilin particles and the intensity of the particles of NPAT and Coilin were calculated. The density shows the degree to which dots overlap with others. Signal intensities were evaluated using more than 200 nuclei for each condition. Wild-type, n = 308; EAF1 mutant, n = 204 in a, and wild-type, n = 278; MED26 mutant, n = 318 in b. c, d Quantification of the number and intensity of Coilin particles. All detected Coilin particles in EAF1-mutant cells (EAF1-mutant) (c) or MED26-mutant cells (MED26-mutant) (d) are plotted. NPAT-associating Coilin particles are indicated by black dots and free Coilin particles are indicated by white dots. Quantification was performed using 300 (c) or 200 (d) Coilin particles. e Super-resolution images showing relative positions of HLBs, Mediator, and CBs. Wild-type cells (WT, upper) and EAF1-mutant cells (EAF1 mut, lower) were fixed with paraformaldehyde, and triple immunofluorescence staining for NPAT (green), MED26 (red), and Coilin (gray) was performed. Enlarged images for representative particles are shown in the lower part of each image. f Calculation of the distance between HLBs and CBs. Immunofluorescence staining for NPAT and Coilin was performed with wild-type and EAF1-mutant HEK293T cells. For Coilin-associated NPAT particles, the distance between the center of the NPAT particle and Coilin particle was measured, and the distance between each associating particle is displayed as boxplots. The center line of each boxplot represents the median. Upper and lower fences of each boxplot represent upper and lower quartiles, respectively. Wild-type, n = 465 particles from 301 nuclei; EAF1 mutant, n = 145 particles from 354 nuclei. Source data are provided as a Source Data file.