Fig. 4: Co-condensation of NONO-TFE3 and PSPC1 is essential for tRCC growth.

a Schematic showing TurboID-based proximity biotin labeling to identify potential interacting proteins for NONO-TFE3 or NONO-TFE3-4A mutant. b Volcano plot showing proteins preferentially interacting with NONO-TFE3 over NONO-TFE3-4A (logFC > 1 or < −1 and p < 0.05, two-sided, unpaired Student’s t-test). c The Gene Ontology (GO) analysis on the proteins that interact with NONO-TFE3, but not NONO-TFEF3-4A mutant (red dots in Fig. 4b). Top 5 enriched categories are listed. d Validation of NONO-TFE3 interaction with PSPC1 or SFPQ by co-immunoprecipitation (Co-IP) in UOK109 KI cells. (n = 3 independent biological replicates). e Schematic representation of the CRISPR screening process. Created in BioRender. Suris, A. (https://BioRender.com/zonrc7c). f Venn diagram showing the candidates number identified from the CRISPR screening with UOK109 and 786-O cell lines. g Plot showing the UOK109 cell specific (red dots), 786-O specific (black dots), and shared (green dots) candidates identified from the CRISPR screening. h Representative images (left) and line profile analysis (right) of endogenous NONO-TFE3 (GFP labeled) and mCherry (mCh) labeled PSPC1 co-condensation droplet in UOK109 KI cells. mCherry (mCh) alone was used as control. n = 3 independent biological replicates; Scale bar, 10 µm. i In vitro co-condensation assay using recombinant proteins of mCherry-tagged NONO-TFE3 and GFP-tagged PSPC1. Scale bar, 10 µm. n = 4 independent biological replicates. j Representative images (left) and quantification (right) of clone formation assay results for UOK109 or 786-O cells expressing the indicated shRNAs. (n = 3 independent biological replicates; one-way ANOVA with Tukey’s post-hoc test). Data are shown as mean ± SD. Source data are provided as a Source Data file.