Fig. 3: Tudor domains and Arginine 207 are necessary for SGF29 phase separation.

a Protein sequence and disorder prediction (PONDR) of the SGF29. The number on the top represents the position of amino acids (aa). b Schematic diagram for EGFP-SGF29 truncation mutants. c Immunofluorescence images of EGFP-SGF29 truncation mutants in HEK293T cells. Left, representative immunofluorescence images. Right, quantification for the condensate numbers of EGFP-SGF29 truncation mutants in HEK293T cells. Data are shown as means ± SEM. n = 50 HEK293T cells. Scale bars, 10 μm. ns, not significant; ***P < 0.001 (t-test). d Coomassie blue staining of purified recombinant SGF29-(54-293) after being resolved on SDS-PAGE. e Formation of phase-separated condensates of purified recombinant SGF29 (54–293aa) forms phase-separated condensates at the indicated concentrations. Left, representative phase images for SGF29-(54–293) droplets. Scale bars, 50 μm. Right, quantification for the turbidity of SGF29-(54–293) droplet. Data are presented as the mean ± SEM. n = 3 biological replicates; ***P < 0.001 (t-test). f The PONDR and the crystal structure of SGF29. The white arrowheads indicate the position of Arg 207 residue in the crystal structure of SGF29. g Subcellular fractionation of exogenous EGFP-SGF29 in the cytoplasmic (Cyt), nuclear soluble (Nuc) and chromatin-associated (Chr) fractions in EP WT hMPCs (P5, young hMPCs) transduced with lentiviruses expressing EGFP-SGF29-WT (WT) or EGFP-SGF29-R207P (R207P). β-Tubulin, TAP1, and H4 were used as the loading control, respectively. h Representative images of hMPCs transduced with lentiviruses expressing either EGFP-SGF29-WT (WT) or EGFP-SGF29-R207P (R207P). Scale bars, 10 μm. i Quantification of SGF29 puncta number per cell in hMPCs transduced with lentiviruses expressing either EGFP-SGF29-WT (WT) or EGFP-SGF29-R207P (R207P). n = 50 hMPCs. Data are presented as the mean ± SEM. ***P < 0.001 (t-test). j Representative time-lapse FRAP images acquired in hMPCs transduced with lentiviruses expressing either EGFP-SGF29-WT (WT) or SGF29-R207P (R207P) with magnified insets showing the pre-bleach and recovery signals of EGFP-SGF29-WT (WT), EGFP-SGF29-R207P (R207P), respectively. Scale bars, 10 μm and 2.5 μm (zoomed-in image). k The curve showing the quantification of fluorescence intensity in FRAP recovery assay indicated in Fig. 3j and Supplementary Fig. S5c. EGFP-RPB1 was used as the slow recovery control. n = 7 hMPCs. **P < 0.01 (t-test).