Extended Data Fig. 11: UTY is a weaker tumour suppressor than UTX due to its altered physical property of condensates. | Nature

Extended Data Fig. 11: UTY is a weaker tumour suppressor than UTX due to its altered physical property of condensates.

From: UTX condensation underlies its tumour-suppressive activity

Extended Data Fig. 11

a. Immunoblotting by indicated antibody of total lysates from THP-1 cells electroporated with empty vector or indicated Myc-tagged UTX WT or mutants. b. Amino acid sequence alignment of UTX and UTY cIDR. Middle row shows identical residues (by letter) and conservative mutations (by “+”). Y and F are labeled with green box. Negatively charged residues (E and D) are in red font, and positively charged residues (R and K) are in blue font. Charged residues that are unique in UTX or UTY cIDR are bold and underlined. c. Net charge per residue for cIDR of UTX and UTY, determined by CIDER. d. Images of Ni resin after binding with bacterial lysates during purification of indicated proteins. Red arrows point to the unusual aggregation of the resin bound to UTY cIDR. e. Coomassie blue staining image of purified UTX cIDR and UTY cIDR with indicated fluorescence tag. A non-specific band is indicated by the asterisk. f. Indicated proteins all at 30 µM protein and in 150 mM NaCl were subjected to centrifugation. The unspun mixture (“mixed”), supernatant (“super”), and pellets were resolved on SDS-PAGE gel followed with coomassie blue staining. Partition Percentage is plotted (right) as described in Methods as mean ± SD of 3 repeats. ***P<0.001 by two-sided t-test. g. Fluorescence and DIC images of mCherry-UTX cIDR or mCherry-UTY cIDR at indicated concentrations and time points after dilution into the condensation buffer. h. Fluorescence microscopy images of mEGFP-UTX cIDR or mEGFP-UTY cIDR at increasing concentrations of protein and NaCl. A shorter exposure time was used for all images of 20 µM protein. i. Fluorescence Image and line carpet of mEGFP-UTX cIDR and mEGFP-UTY cIDR droplets. j. Line RICS autocorrelation curves of 50 µM mEGFP-UTX cIDR and mEGFP-UTY cIDR at early (< or = 100 min) or late (> 170 min) time of phase separation after dilution into the condensation buffer, shown as mean ± SD. n UTX early = 8, n UTY early = 9, n UTX late = 8, n UTY late = 9 droplets. k. Representative fluorescence microscopy images for FRAP assays of UTX or UTX-UTYIDR fused to EGFP in nuclei following transfection into 293T cells. The photobleached focus was boxed and also amplified in the images on the right. Photobleached at time 0.

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