Fig. 3: RNA-dependent condensation of NONO-TFE3 supports tRCC growth.

a Representative images (left) and quantification (right) of droplet formation in UOK109 cells transfected with NONO-TFE3-GFP or NLS-TFE3 S321A. NLS: nuclear localization signal. (n = 8 cells from 3 independent biological replicates; two-sided unpaired Student’s t-test). Scale bar, 10 µm. b Representative images (left) and normalized fluorescence signals (right) of endogenous NONO-TFE3-GFP during FRAP assay in UOK109 KI cells (n = 3 cells). The red arrow: photobleaching starting time point. Scale bar, 10 µm. c Representative live-cell imaging showing fusion events of endogenous NONO-TFE3-GFP over time (red box). n = 4 independent biological replicates. Scale bar, 5 µm. d Domain structure (top) and intrinsically disordered tendency (bottom) of NONO-TFE3 calculated by Predictor of Natural Disordered Regions (PONDR). A score of over 0.5 indicates predicted intrinsically disordered regions (IDRs). e Representative images (left) and quantification (right) of droplet formation in UOK109 parental cells transfected with GFP-fused NONO-TFE3 truncation variants. (n = 8 cells from 3 independent biological replicates; one-way ANOVA with Tukey’s post-hoc test). Scale bar, 10 µm. f The LacI-LacO array for multivalent homotypic interaction detection between NONO-TFE3 and its truncations. NONO-TFE3 truncates-LacI are labeled with GFP, and NONO-TFE3 WT is labeled with mCherry. If homotypic interaction exists, both GFP- and mCherry-labeled proteins will form puncta at endogenous LacO arrays. g Representative images (left) and quantification (right) of puncta formation in U2OS cells containing LacI-LacO array transfected with the indicated NONO-TFE3 truncation variants. The LacO array locus is circled and images representing the zoom-in views of LacO array loci are shown (white box). (n = 6 cells from 3 independent biological replicates; one-way ANOVA with Tukey’s post-hoc test). Scale bar, 10 µm. h Tumor growth curves of UOK109 KI xenografts transduced with shRNA against NONO-TFE3 and rescued with the indicated constructs. (n = 10 mice; one-way ANOVA with Tukey’s post-hoc test). i Representative images of isolated tumors (left) and quantification (right) of tumor weights at day 24 of in vivo models mentioned in h. (n = 10 mice; one-way ANOVA with Tukey’s post-hoc test). Data are shown as mean ± SD. Source data are provided as a Source Data file.