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. 2021 Mar 19;22(5):e51740. doi: 10.15252/embr.202051740

Figure EV1. Inhibition of Hsp90 delays SG dissolution independently of Hsp70. Related to Fig 1 .

Figure EV1

  • A
    Kinetics of SG dissolution of living cells in absence and presence of ammonium chloride. G3BP2‐GFP HeLa‐Kyoto cells were treated with sodium arsenite (50 µM) for 45 min. Then, cells were allowed to recover in drug‐free medium (recovery control) or in presence of ammonium chloride (NH4Cl, 20 mM). Images were taken over a time period of 4 h every 10 min. Dashed lines = 95% confidence intervals. Number of cells counted: 193 (Recovery Control); 104 (NH4Cl, 20 mM).
  • B
    G3BP2‐GFP HeLa‐Kyoto cells were either left untreated (control) or treated with GA (5 µM), 17AAG (5 µM) or VER (40 µM) for 4 h. Cells were fixed, and the percentage of cells with SGs was counted. n = 3 independent experiments, ± s.e.m.; 126–185 cells counted/sample. n.s.: non‐significant (one‐way ANOVA).
  • C, D
    Kinetics of SG dissolution of living HEK293 cells that express the SG‐resident protein PABP endogenously tagged with the fluorescent probe Dendra2 (PABPC1‐Dendra2). PABPC1‐Dendra2 HEK293K cells were treated with sodium arsenite (100 µM) for 45 min. Then, cells were allowed to recover in drug‐free medium (recovery control) or in presence of ammonium chloride (NH4Cl, 20 mM), GA (5 µM), 17AAG (5 µM), or VER (40 µM). Images were taken over a time period of 4 h every 10 min. Dashed lines = 95% confidence intervals. Number of cells counted: 299 (recovery control); 160 (NH4Cl, 20 mM); 361 (GA 5 µM); 193 (17AAG 5 µM); and 259 (VER 40 µM).
  • E
    Confocal microscopy showing that Hsp90 does not colocalize with DRiPs. HeLa cells were treated with OP‐puro (25 µM) for 1 h. DRiPs were visualized by click chemistry, while Hsc70, Hsp70, Hsp90 α, and β were visualized by immunostaining. Scale bar is 10 µm.
  • F
    Quantitation of DRiP enrichment in SGs. Automated imaging and SG segmentation are based on G3BP signal. Data are presented as histogram. HeLa cells were treated with HS at 43.5°C for 1 h alone or in presence of GA (5 µM), 17AAG (5 µM), or VER (40 µM). Number of SGs segmented: 2,198 (control); 3,056 (VER); 1,484 (GA); 3,220 (17AAG); P < 10−10 (one‐way ANOVA).
  • G, H
    HeLa cells stably expressing V5‐tagged inducible Hsp70 under the control of tetracycline (Flp‐In) were cultured in absence or presence of tetracycline (V5‐HSP70 OFF and V5‐HSP70 ON, respectively). (G) Protein extracts were prepared 24 h after treatment, and Hsp70 levels were analyzed by immunoblotting. TUBA4A was used as loading control. (H) Cells were treated with sodium arsenite (500 µM) for 45 min, followed by recovery in drug‐free medium (control) or in presence of VER (40 µM), GA (5 µM) or 17AAG (5 µM) for 90 min. Cells were then fixed, and the percentage of cells with persisting SGs was counted. n = 3 independent experiments, ± s.e.m.; 115‐378 cells counted/sample; P = n.s.: non‐significant (one‐way ANOVA).
  • I
    HeLa cells were either left untreated or treated with VER (40 µM), GA (5 µM), or 17AAG (5 µM) for 4 h or 24 h, respectively. Cells were fixed and stained for endogenous DCP1A, a marker of P‐bodies. Nucleic acid was stained with DAPI. Quantitation of the percentage of cells with P‐bodies after 4 or 24 h of treatment is shown. n = 3 independent experiments, ± s.e.m.; 4 h: 104–124 cells counted/sample, P = 0.02; 24 h: 111–178 cells counted/sample, P < 10−7 (one‐way ANOVA).
  • J
    G3BP2‐GFP HeLa‐Kyoto cells were lipofected with a cDNA encoding for mRFP‐DCP1A. 24 h post‐transfection, cells were treated with sodium arsenite (50 µM) for 45 min. Then, cells were allowed to recover in drug‐free medium (recovery control) or in presence of GA (5 µM), 17AAG (5 µM), or VER (40 µM). Images were taken over a time period of 2 h every 10 min. Representative images of GFP‐G3BP2 SGs and mRFP‐DCP1A P‐bodies at 0, 30, 60, 90, and 120 min of recovery time are shown. Scale bar is 10 µm.