Validated p38α and mTOR inhibitors discovered in the screen, characterization of H₂O₂-induced INAVA puncta as biomolecular condensates, and schematic of the screen for puncta inhibitors. (A–C) Dose–response curves of all (A) p38α inhibitors, (B) mTOR inhibitors, and (C) translation inhibitors validated from the IL-1β–induced INAVA puncta inhibition screen. Images were taken by high-content imaging at 20× magnification at four positions/well, mean ± SEM, n = 3. (D) Representative images from dose–response experiments (p38α inhibitors [c–m]: p38α inhibitor VIII, PD169316, RWJ-67657, LY2228820, VX702, SB202190, SB239063, PH-797804, GW856553X, SB242235, SB220025; mTOR inhibitors [n–q]: GDC-0980, BEZ235, KU0063794, PI-103). Hoechst (nuclei). Scale bar = 10 μm. (E) HCT8-INAVA-GFP cells were pretreated with INK128 (10 μM) or SB203580 (10 μM) for 30 min followed by treatment with IL-1β for 90 min. Cells were harvested and analyzed by Western blotting. Anti-HA detects INAVA-GFP, and actin was used as loading control. (F) HCT8-INAVA-GFP cells were treated with 3% and 5% 1,6-hexanediol (HD) for 5 and 15 min and were analyzed for INAVA-GFP puncta, mean ± SD. (G) Same as F but the time point at 30 min with 3% 1,6-HD, n = 3. (H and I) NF-κΒ luciferase reporter assay with (H) p38α inhibitor SB203580 or (I) mTOR inhibitor INK128. HEK293T cells transfected with empty vector (EV) or INAVA, NF-κΒ firefly luciferase and SV40-R. reniformis were pretreated with SB203580 or INK128 for 1 h and then treated with IL-1β (10 ng/ml) for 4 h and assayed for luminescence. Each point represents average luminescence for all replicates within each experiment, N = 49 per condition, four independent experiments performed, mean ± SD. (J) HCT8-INAVA-GFP cells expressing doxycycline-inducible myc-TRAF6. Cells were treated overnight with doxycycline (1 μg/ml) and then treated with IL-1β (10 ng/ml) for 60 min. Cells were fixed, stained with α-myc to detect myc-TRAF6 and processed by spinning disk confocal imaging. Scale bar = 10 μm. (K) FRAP of H₂O₂-induced INAVA-GFP “young” (45 min; n = 6) and “old” (120 min; n = 5) puncta in HCT8-INAVA-GFP cells. Bleaching was performed at the indicated time point (arrow), and recovery was allowed to occur at 37°C in 5% CO₂, mean ± SEM. (L) Velocity tracking of H₂O₂-induced INAVA-GFP puncta formed at different time points, mean ± SEM, n = 36. (M) Schematic of screen to identify inhibitors of H₂O₂-induced INAVA-GFP puncta (left panel) in HCT8 cells. Shown are ranked Z-scores of hits (right panel) and relative ranking of select compounds of interest: inhibitors of protein translation (green), MAPK p38α (red), or mTOR (blue). Frequencies of distribution of negative controls are displayed to the right of the graph. (N) HCT8-INAVA-GFP cells were pretreated with cycloheximide (CHX; 10 μM) or MELK inhibitor OTSSP167 (10 μM) for 30 min followed by IL-1β (10 ng/ml) for 1 h. Cells were harvested and analyzed for Western blotting. Anti-HA measures HA tag present in INAVA-GFP, and actin was used as a loading control. ****, P < 0.0001. The unit of measure for the Western blots is kilodaltons.