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

Figure EV5. Hsp90 couples SG dissolution and translation restoration via DYRK3.

Figure EV5

  • A
    HeLa‐Kyoto cells were left untreated or treated with sodium arsenite (50 µM; A) for 45 min to induce SGs, followed by recovery in drug‐free medium or in presence of LY294002 (50 µM) for 4 h. Where indicated cells were only treated with LY294002 (50 µM) for 4 h. Expression levels of total and phosphorylated p70 S6K and PRAS40 were investigated by immunoblotting. TUBA4A was used as loading control.
  • B
    HeLa‐Kyoto cells were treated as described above except that Wortmannin (200 nM) was used instead of LY294002 (50 µM).
  • C, D
    Confocal microscopy on G3BP1‐mCherry HeLa‐Kyoto cells overexpressing myc‐Raptor were left untreated or treated with sodium arsenite (ARS; 50 µM) for 45 min to induce SGs, followed by recovery in drug‐free medium or in presence of GA (5 µM), 17AAG (5 µM, high or 0.5 µM, low) for 2 h. Scale bar is 10 µm.
  • E
    HeLa‐Kyoto cells were treated as described above, and protein extract was prepared at the indicated time points. Expression levels of p70 S6K, PRAS40, and 4E‐BP1, total and phosphorylated forms, were investigated by immunoblotting. TUBA4A was used as loading control.
  • F
    Impact of treatments of GA (5 µM), 17AAG (5 µM, high or 0.5 µM, low) for 4 h on total and phosphorylated p70 S6K, PRAS40, and 4E‐BP1.
  • G
    HeLa‐Kyoto cells were treated with sodium arsenite (50 µM) for 45 min to induce SGs, followed by recovery in drug‐free medium or in presence of GA (5 µM), 17AAG (5 µM, high or 0.5 µM, low) for 1 and 4 h. Where indicated, 15 min prior to protein extraction, puromycin (5 µg/ml) was added to the medium. The total levels of puromycilated proteins were investigated by immunoblotting. TUBA4A was used as loading control.
  • H
    Working model showing that Hsp90 inhibition impairs the activation of the mTORC1 pathway via inhibition of DYRK3‐mediated disassembly of SGs (SG‐dependent) and inhibition of Akt (SG‐independent).

Data information: Related to Movies EV8 and EV9.

Source data are available online for this figure.