Figure S2.
Characterization of INAVA puncta inducers. (A and B) Dose–response curves of (A) HSP90 inhibitors or (B) proteasome inhibitors. Images were taken by high-content imaging at 20× magnification with four positions/well, mean ± SEM, n = 3. (C) Representative images from A and B. Scale bar = 10 μm. (D) Representative images from ROS inducers identified in the INAVA puncta inducer screen. Scale bar = 10 μm. (E) HCT8 cells stably expressing only GFP were treated with corresponding compounds for 90 min, fixed, and imaged. Scale bar = 40 μm. (F) HCT8-INAVA-GFP cells treated with IL-1β (10 ng/ml), H2O2 (1 mM), or ganetespib (1 μM) for the early and late time points indicated. Cells were fixed and stained with vimentin. Scale bar = 20 μM. (G) INAVA protein–protein interactors annotated in BioGRID (https://thebiogrid.org). Dashed circles highlight interactors, including cytohesin family members (blue) and multisubunit ubiquitin E3 complexes (green). The multisubunit dynactin complex (red) involved in the aggresome pathway was also identified. (H and I) HCT8-INAVA-GFP cells were treated with (H) IL-1β (10 ng/ml), ganetespib (1 μM), MG132 (10 μM), or (I) H2O2 (1 mM) for 90 min. Cells were fixed and stained with AMYLO-GLO. Scale bar = 20 μM. (J) HCT8-INAVA-GFP cells were treated with IL-1β (10 ng/ml) or H2O2 (1 mM) for 90 min and stained with EDC4 (left panel). Fluorescent line scans (arrow) of INAVA (green) and EDC4 (red) are displayed (right panel). Scale bar = 10 μm. (K) HCT8-INAVA-GFP cells were treated with IL-1β (10 ng/ml), ganetespib (1 μM), or H2O2 (1 mM) for 90 min and stained with HDAC6. Scale bar = 20 μm. (L) Pulse-chase experiments in HCT8 INAVA-GFP cells treated with IL-1β (10 ng/ml), H2O2 (1 mM), or ganetespib (1 μM) for 30 min. Following treatment, cells were washed with complete media, chased, and analyzed at the time points indicated, mean ± SEM, n = 2. (M) HCT8-INAVA-GFP cells were treated with condensate inducers as above, including MG132 (10 μM) at the indicated time points. 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. (N) Same as D but treated with ganetespib (1 μM and 300 nM). Samples were collected at 1.5 h and 20 h for Western blot analysis. The unit of measure for the Western blots is kilodaltons.

Characterization of INAVA puncta inducers. (A and B) Dose–response curves of (A) HSP90 inhibitors or (B) proteasome inhibitors. Images were taken by high-content imaging at 20× magnification with four positions/well, mean ± SEM, n = 3. (C) Representative images from A and B. Scale bar = 10 μm. (D) Representative images from ROS inducers identified in the INAVA puncta inducer screen. Scale bar = 10 μm. (E) HCT8 cells stably expressing only GFP were treated with corresponding compounds for 90 min, fixed, and imaged. Scale bar = 40 μm. (F) HCT8-INAVA-GFP cells treated with IL-1β (10 ng/ml), H2O2 (1 mM), or ganetespib (1 μM) for the early and late time points indicated. Cells were fixed and stained with vimentin. Scale bar = 20 μM. (G) INAVA protein–protein interactors annotated in BioGRID (https://thebiogrid.org). Dashed circles highlight interactors, including cytohesin family members (blue) and multisubunit ubiquitin E3 complexes (green). The multisubunit dynactin complex (red) involved in the aggresome pathway was also identified. (H and I) HCT8-INAVA-GFP cells were treated with (H) IL-1β (10 ng/ml), ganetespib (1 μM), MG132 (10 μM), or (I) H2O2 (1 mM) for 90 min. Cells were fixed and stained with AMYLO-GLO. Scale bar = 20 μM. (J) HCT8-INAVA-GFP cells were treated with IL-1β (10 ng/ml) or H2O2 (1 mM) for 90 min and stained with EDC4 (left panel). Fluorescent line scans (arrow) of INAVA (green) and EDC4 (red) are displayed (right panel). Scale bar = 10 μm. (K) HCT8-INAVA-GFP cells were treated with IL-1β (10 ng/ml), ganetespib (1 μM), or H2O2 (1 mM) for 90 min and stained with HDAC6. Scale bar = 20 μm. (L) Pulse-chase experiments in HCT8 INAVA-GFP cells treated with IL-1β (10 ng/ml), H2O2 (1 mM), or ganetespib (1 μM) for 30 min. Following treatment, cells were washed with complete media, chased, and analyzed at the time points indicated, mean ± SEM, n = 2. (M) HCT8-INAVA-GFP cells were treated with condensate inducers as above, including MG132 (10 μM) at the indicated time points. 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. (N) Same as D but treated with ganetespib (1 μM and 300 nM). Samples were collected at 1.5 h and 20 h for Western blot analysis. The unit of measure for the Western blots is kilodaltons.

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