Figure 6. Heat shock chaperones regulate A-bodies disaggregation.

(A) Amyloidogenesis is rapid and reversible. MCF-7 and PC-3 cells exposed to acidosis or heat shock and allowed to recover (times indicated) were stained with Congo red. The proportion of cells containing Congo red-positive structures was assayed relative to Hoechst-positive nuclei. (B) Heat shock proteins mediate the solubilization of A-body components. Insoluble proteins were extracted from untreated, acidotic or recovering MCF-7 cells treated with the protein synthesis inhibitor cycloheximide (Chx) or PDI (16F16), GRP94 (EGCG), Hsp70 (VER155008) or Hsp90 (17-AAG) inhibitors. POLD1, cdk1, GAPDH and Histone H3 were detected by western blot. (C) Heat shock proteins are associated with the A-bodies during recovery. Table summarizing data in Figure S6B. (D) Heat shock proteins disaggregate the A-bodies. MCF-7 cells were exposed to acidotic (left panel) or heat shock (right panel) conditions for 3 hours, then returned to normal growth conditions for 2 or 4 hours in the presence 16F16, EGCG, VER, AAG or Wortmannin. The proportion of Congo red-positive cells was determined as above. (E) Congo red stained MCF-7 cells allowed to recover for 2 or 4 hours from a 3 hour acidosis or heat shock exposure in the presence or absence of VER155008. (F) Hsp70 activity enhances β-amyloid release during recovery. β-amyloid-GFP-expressing MCF-7 cells were allowed to recover for 4 hours from a 3 hour heat shock exposure in the presence or absence of VER or Chx. Western blots of insoluble fractionation for β-amyloid-GFP and Histone H3 are included (lower left). Results are means and SEM (n=4). Significance was measured by Student’s t-test; *p < 0.01. Dashed circles represent nuclei. White scale bars represents 20µm. See also Figure S6.