Fig 1: Time course imaging reveals that punctate bodies are dynamic, shown here for HeLa cells in purine-depleted medium transfected with PAICS-RFP grown in two replicate 4 hour time series.Panels (A) and (D) show the time zero condition; panels (B) and (E) show the same cells as in (A) and (D), respectively, following two hours of growth in the same medium; panels (C) and (F) show the same cells following two hours of growth after exchanging the growth medium to purine-rich. Cells marked by * display formation of punctate bodies over the time series, while cells marked by p display variable dynamics of punctate bodies. The # sign marks cells with punctate bodies that die over the course of the series; the cell marked by d dies in the absence of punctate bodies. Cell death was determined by marked cell shrinkage and membrane blebbing, detected by differential interference contrast (DIC) microscopy, as in panel (C-DIC), accompanied by markedly increased cellular fluorescence, easily distinguishable from flat healthy cells and mitotic cells (one is marked by m in panels (C) and (C-DIC)). Notably, punctate bodies are detectable in both purine-poor and rich media, with some forming even after the shift into purine-rich medium, as for the cell marked * in (E-F).
Fig 2: The cell stressor hydrogen peroxide (H2O2) strongly induced purine biosynthetic enzyme punctate bodies regardless of hypoxanthine (Hx) presence.Base medium is DMEM supplemented with 10% FBS. As indicated, medium was also supplemented with 1 mM H2O2 and/or 35 µM Hx as described in Methods. For HEK293 PPAT-EGFP cells, n = 4419, 2652, 3088, 3182 cells per bar. For HEK293 PAICS-RFP cells, n = 2970, 1944, 1880, 1760. For HEK293T PPAT-EGFP cells, n = 4537, 2267, 2411, 2947. For HEK293T PAICS-RFP cells, n = 4612, 3660, 4211, 3760. Bars indicate average +/- 1 s. d. across at least 3 replicates.
Fig 3: Purine biosynthesis enzymes only rarely co-localized in intracellular bodies, showing even partial co-localization in no more than 4% of co-transfected cells as assayed in HeLa cells and quantified in Table S1.The top row shows an example of partial but minimal co-localization of FGAMS-EGFP and PAICS-RFP bodies. The middle row shows an example of non-colocalizing FGAMS-EGFP and ADSL-RFP bodies. The bottom row shows an example of a more typical case, non-co-localization due to the formation of bodies by only one protein in doubly-transfected cells, as shown here for PPAT-EGFP and PAICS-RFP.
Fig 4: Co-expressed HSP70 and HSP90 chaperones marked purine biosynthesis bodies.Intracellular bodies formed by (A) PPAT-EGFP, (D) FGAMS-EGFP and (G) PAICS-RFP co-localized with co-transfected (B,E) HSP70-RFP or (H) HSP70-GFP, shown here in HeLa cells. (C, F and I) show merged images. (J-L) Intracellular bodies were also often observed to co-localize with co-transfected HSP90, shown here with PPAT-EGFP.
Fig 5: Formation of intracellular bodies in HEK293T cells scaled with DNA transfected.Among successfully transfected cells, the fraction of the cell population exhibiting PAICS-RFP puncta correlated strongly with the quantity of plasmid DNA transfected. Bars indicate average +/- 1 s. d. across at least 3 replicates, n = 498, 627, and 591 cells, respectively.
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