FIG 6.

Maintenance of NM-HA aggregates over multiple cell divisions. (A) Experimental setup to study aggregate maintenance over several cell passages. In a given population, the number of cells stably expressing the transgene was determined for each passage. These cells were then analyzed for NM-HA aggregates. (B) Percentage of cells expressing wild-type or mutant NM-HA that harbor aggregates over multiple cell divisions. Cell populations were exposed to 10 μM untagged recombinant NM fibrils (monomer concentration) and subsequently passaged every 2 to 3 days. Cells with aggregates were determined by automated confocal microscopy analysis (n = 3 biological replicates). Bars represent mean values ± SEM. P, passage. At least 7,900 cells were analyzed per cell population per passage. Proliferation doubling time for N2a NM-HA cells is approximately 13 h (data not shown). This corresponds to approximately 51 doublings within 13 passages. (C) Fold change of the probability for NM-HA mutant cell populations to lose aggregate-harboring cells over time compared to cells expressing full-length NM-HA. >0 denotes lower mitotic stability than NM-HA, and <0 denotes higher mitotic stability than NM-HA. Bars represent mean values, and error bars represent the upper limit for the confidence interval (n = 3). The Cox proportional hazard model was used for statistics (cell count, >12,500; ****, P ≤ 0.0001). (D) Time-lapse analysis of N2a cell lines stably expressing NM-GFP full-length, Δ 1-39, or Δ 75-123. Cells were exposed to 10 μM untagged recombinant NM fibrils for 24 h (monomer concentration). Two passages postinduction, cells were subjected to live cell imaging for a total of 15 h. Scale bar, 10 μm.