Fig 1: MS023 treatment induces LLPS of candidate RBPs. (A) Tendency of MS023-modulated RBPs to undergo LLPS. Among the 77 proteins up-regulated in the interface fraction upon MS023, 43 (56%) were also annotated as proteins undergoing phase separation in at least one of the two PhaSepDB (http://db.phasep.pro/) and RNA Granules DB (http://rnagranuledb.lunenfeld.ca/) databases. Among them, 16% were annotated in both databases, 33% were annotated only in PhaSepDB and 51% were annotated only in RNA Granules DB. (B) Immunofluorescence (IF) analysis of LDHB in basal condition (DMSO) and in response to different treatments. Representative IF image shows LDHB protein subcellular localization in HeLa cells treated with the following compounds: DMSO, 10 µM MS023 and 5 µM GSK591 for 48 h; 10 µM MS023 for 48 h, followed by 10 min treatment with 5% 1,6-Hexanediol; 400 µM NaAsO2 for 30 min, or 400 µM NaAsO2 for 30 min followed by 10 min-treatment with 5% 1,6-Hexanediol. Immunostaining of RNA was performed with the Click-iT™ RNA Alexa Fluor™ 594 Imaging Kit. DAPI staining was used for nuclei visualization (DNA). G3BP1 staining was used as positive control for SGs formation. DAPI, LDHB, G3BP1 and RNA staining and the respective merged images are displayed. Images were taken by SP8 confocal microscopy using a 60× oil objective, and a scale bar of 25 µM are included in the merged figure. Arrows indicate co-localization of target RBP, G3BP1 and RNA. (C) Bar-graph representation of the percentage of cells with stress granules. The bar-graph shows the percentage of cells with at least 1 G3BP1-positive stress granule (SG) for the different conditions; all the treatments were normalized over DMSO. (D) Bar-graph representation of the percentage of LDHB-G3BP1 co-localization. The image describes the percentage of co-localization between G3BP1 and LDHB in the SGs in each condition. All the treatment were normalized over the DMSO. Statistical significance was calculated by Student’t Test (* = p <0.05).