Fig. 5: JMJD3 undergoes LLPS in vitro and in vivo.
a mEGFP and mEGFP–JMJD3 proteins were analyzed using droplet-formation assays in nuclear extracts at room temperature with 150 mM NaCl. Quantifications of the number of droplets per frame, circularity, convexity and aspect ratio (AR) are displayed. Data are the mean ± SEM. Boxes comprise values from Q1 to Q3 of the dataset; line corresponds to median value; whiskers show the data range (from min. to max. values within dataset). ***p < 0.001 (Student’s t test, p = 1.09982E-06). Droplets in 5 fields in each group from three biologically independent experiments were quantified, n = 150. Scale bar, 5 μm. b Confocal microscopy images of HEK293T cells transfected with mEGFP-JMJD3. Quantifications of the intensity of JMJD3 puncta are shown on the right. Data show the mean ± SEM. Boxes comprise values from Q1 to Q3 of the dataset; line corresponds to median value; whiskers show the data range. ***p < 0.001 (Student’s t test, p = 9.98785E-06 and p = 7.15724E-17). n = 50 transfected cells in each group were quantified; Images are representative of 3 biologically independent experiments. Scale bar, 5 μm. Western blot displays the levels of overexpressed JMJD3. c HEK293T cells were transfected with 0.05 ug mEGFP–JMJD3, treated with 6% 1,6-HD for 5 min and imaged at 60 and 120 s. Nuclei were visualized with DAPI (blue). Quantification of the nuclear puncta per cell is shown on the right. Data are the mean ± SEM. *p < 0.05 (Student’s t test, p = 0.0182428 and p = 0.01162199). n = 130 transfected cells were quantified; Images are representative of three biologically independent experiments. Scale bar, 5 μm. d NSCs and HEK293T cells were fixed, and endogenous JMJD3 was visualized by immunostaining assay. The images are representative of three biologically independent experiments. Scale bar, 5 μm. e FRAP assay in HEK293T cells expressing 0.05ug of mEGFP–JMJD3. Images are representative of three biological replicates. Quantification shows the curve fit results of FRAP data for mEGFP-JMJD3 to a double-exponential smoothing (R2 = 1), where bleaching events occurs at t = 0 s. Data are plot as background-subtracted and normalized mean (n = 27 cells). Scale bar, 5 μm. Source data are provided as a Source Data file.
