Extended Data Fig. 3: Tumour suppressive activity of UTX can be maintained by replacing its cIDR with unrelated protein IDRs. | Nature

Extended Data Fig. 3: Tumour suppressive activity of UTX can be maintained by replacing its cIDR with unrelated protein IDRs.

From: UTX condensation underlies its tumour-suppressive activity

Extended Data Fig. 3

a. Immunoblotting by anti-UTX or GAPDH of total lysates from THP-1 cells electroporated with empty vector or indicated UTX WT or mutants. b. Immunoblotting by anti-UTX (top) or GAPDH (bottom) of total lysates from THP-1 or MiaPaca2 cells that were transduced with empty vector or indicated UTX WT or mutants and induced with doxycycline. c. Confocal images of 293T cells transfected with indicated UTX chimeric constructs all fused to EGFP. d. Growth curves of THP-1 cells electroporated with indicated vector or UTX constructs, shown as average ± SD. n = 3 independent experiments. e. Representative results of colony formation assay of THP-1 cells transduced with indicated UTX WT or mutant construct. Bottom left, the number of colonies are plotted as mean ± SD (n = 3 independent experiments). Bottom right, the size of colonies are plotted as mean ± SD. n = 100 colonies for each except 73 colonies for UTX-eIFIDR. f. Kaplan Meier Curves for survival of the animals grafted with THP-1 cells stably transduced with indicated vector or UTX constructs. P = 0.0024 for WT vs. ΔcIDR, and 0.015 for ΔcIDR vs. UTX-eIFIDR. n = 8 mice each, except n = 10 for Vector. g. Representative crystal violet staining images of MiaPaca2 cells transduced with indicated UTX WT or mutant construct. Two thousand cells were seeded and cultured for 7 days in the absence or presence of doxycycline (to induce transgene expression) before staining. Right, staining absorbance for cells with doxycycline are plotted as mean ± SD from n = 3 independent repeats. h. MiaPaca2 cells stably transduced with indicated vector or UTX constructs were grafted into immune-deficient mice. Tumours from day 20 were imaged and their weights are plotted as average ± SD. n = 5 mice each for Vector and UTX-FUS(IDR), or 6 mice each for the others. i. Immunoblotting of total lysates (Input) and anti-FLAG antibody-mediated immunoprecipitation (IP) from 293T cells transfected with empty vector, or FLAG-tagged UTX WT or ΔcIDR, using indicated antibodies. jo. RNA-seq analysis for gene expression in the THP-1 cells transduced with indicated UTX constructs (two samples each). j. Volcano plot. Blue and red dots represent genes that were significantly (adj. P<0.05) up- or down-regulated (over 1.25 fold), respectively, by WT versus vector control. The adjusted P values on y axis were from DESeq2. The detailed gene information is in Supplementary Table 1. k. Gene ontology analysis for the indicated gene clusters from the heat map in Fig. 1j. Red plot shows the significantly enriched gene functions for the 300 genes in the red bracket in Fig. 1j, which were strongly induced by WT and UTX-eIFIDR, but not as strongly by ΔcIDR, and not affected by ΔTPR. Blue shows the significantly enriched gene functions for the 200 genes in the blue bracket in Fig. 1j, which were markedly repressed by WT and UTX-eIFIDR, but not ΔcIDR and ΔTPR. l. Principal component analysis of quantile normalized gene expression (log2 of TPM, pseudo-count=1) using 1,271 DEGs in WT versus vector control. m. Scatter plots for gene expression changes between indicated samples. Black dashed line indicates the linear fit with correlation coefficient (r) labeled. Red and blue dots are genes significantly up- or down-regulated (over 1.25 fold) in WT over vector, respectively, as in j. n. Box plots for gene expression changes between indicated samples for the up- and down-regulated genes (defined in m) in WT over vector. Data are median (horizontal line), 25–75th percentiles (box) and 1.5 times the interquartile range recorded (whiskers). P values are shown for data significantly greater or less than 0 by two-sided one sample t-test. Left, n = 675 genes each; right, n = 596 genes each. o. Heatmap showing relative expression levels of genes that were significantly (adj. P<0.05) up- or down-regulated (1.25 fold each) by WT compared to vector control in the THP-1 cells transduced with vector control, UTX WT or catalytically inactive (MT2) mutant (two samples each). Each row represents a gene. P values by one-way ANOVA followed by Tukey’s post hoc test for all analyses except by two-sided log-rank test for g, two-sided modified Fisher’s exact test for k, and two-sided one sample t-test for n.

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