Figure 1. Uncovering the distinct biophysical properties of the cellular A-bodies.

(A) Physiological amyloidogenesis is rapid and reversible. MCF-7 cells exposed to extracellular acidosis and returned to standard growth conditions, for the indicated times, were stained with Congo red and Hoechst (blue inset). (B) Nuclear foci stain positively with amyloid-specific dyes. Untreated or acidotic MCF-7 cells were stained with Amylo-Glo (blue), Thioflavin S (green) and/or Congo red (red). Selected regions (white box) were expanded below with merged image included (far right panel). Dashed circles represent nuclei. (C) Established proteins are targeted to the A-bodies. MCF-7 cells expressing VHL-GFP or POLD1-GFP were grown under standard, hypoxic/acidotic conditions or recovered for 24 hours post-acidosis treatment. Acidotic cells were stained with Congo red. Selected regions (white box) were expanded (below). (D) A-body targets are reversibly immobilized. VHL-GFP, POLD1-GFP or GFP-B23 transfected MCF-7 cells were treated as above and bleached repeatedly for fluorescent loss in photobleaching. Quantification is presented as the mean relative intensity of at least 5 data sets. (E) Stimuli-specific insolubilization of A-body components. Insoluble proteins (Insol) were extracted from whole cell lysates (WCL) of untreated, acidotic (2 hrs), heat shocked (2 hrs), sodium arsenite (1 hr), cycloheximide (1hr), thapsigargin (8 hrs) or H₂O₂ (8 hrs) treated cells. A-body components; VHL-GFP and endogenous POLD1 or the GAPDH and Histone H3 control proteins were detected by western blot. (F) Stimuli-induced A-bodies are proteinase K resistant. Transmission electron microscopy (TEM) of untreated, heat shocked (1hr) and acidotic (1hr) MCF-7 cells were left undigested or exposed to proteinase K. Proteinase K-resistant nuclear bodies are indicated (yellow arrow). (G) Proteinase K-resistant fibrils possess amyloid-like properties. Heat shocked MCF-7 cells were left undigested or proteinase K-treated prior to TEM visualization. Proteinase K-resistant structures were stained with Congo red or the amyloid fibril conformation-specific antibody OC. (H) Protein insolubilization correlates with A-body assembly and disaggregation. MCF-7 cells were treated as above (A) and insoluble proteins were extracted from WCL. A-body targets/controls were detected as in (E). (I) Inhibition of rIGSRNA transcripts impairs amyloidogenesis. MCF-7 cells stably-expressing control or shRNA against rIGS₂₈RNA or rIGS₂₂RNA were grown in acidosis permissive media or exposed heat shock, respectively, prior to Congo red staining. Dashed circles represent nuclei. White scale bars represents 20mm. Black and white TEM scale box represent 1µM and 0.1mm. See also Figure S1.