(A) A monoclonal HEK293 line stably expressing tau RD(LM)-YFP (hereafter referred to as tau RD) was transduced with tau RD fibrils. At Day 3, cells were diluted sparsely in a 10 cm dish. At Day 12, inclusion-positive colonies were identified and picked, amplifying to confluency in separate 10 cm dishes. At Day 30, cells were re-plated for confocal analysis or harvested for subsequent experiments.
(B) Confocal analysis of morphologically distinct tau RD prion strains. Clone 1 does not contain inclusions. Clone 9 contains nuclear speckles and a small juxtanuclear inclusion. Clone 10 features one very large juxtanuclear inclusion and no nuclear speckles. See for other clones.
(C) Clones 1, 9 and 10 were stained with X-34, an amyloid dye. X-34 staining is only observed in Clone 9 and Clone 10, indicating that the propagated aggregates are amyloids.
(D) SDD-AGE demonstrates that Clone 10 features larger aggregates than Clone 9.
(E) Sedimentation analysis was performed on Clones 1, 9, and 10. Pellet (P) was isolated from supernatant (S) by ultracentrifugation. For Clones 9 and 10, supernatant was loaded at a 3:1 ratio to pellet and total (T) to allow clear detection; Clone 1, a 1:1 ratio. Clone 1 has all tau RD in the supernatant, whereas Clone 9 has almost all tau RD in the pellet. Clone 10 has mixed solubility.
(F) Limited proteolysis (pronase) digests all tau RD in Clone 1, but reveals protease-resistant tau RD peptides between 10 and 13 kDa as well as between 20 and 25 kDa in Clone 9 and 10. Unlike Clone 9, Clone 10 digestion produces a doublet, consistent with a distinct conformation.
(G) A split-luciferase assay reports differential seeding efficiency of tau RD prion strains. A polyclonal HEK293 line expressing both tau RD-CLuc and tau RD-Nluc was transduced with lysate from the three clones. Clone 1 does not seed aggregation. Clone 9 seeds robustly, whereas Clone 10 seeds significantly less. Averages of four separate experiments are shown, each read in quadruplicate 48 hours post-transduction (error bars = S.E.M, * = p<0.05, **** = p<0.0001). See for evidence that differences in cell confluency do not account for differences in luminescence.
(H) Inclusion elimination rates differ between clones. After transduction with lysate from Clone 9 or 10, the percentage of cells containing inclusions was quantified on days 4, 17, and 30 (n=10 fields, each with 150+ cells per condition). Cells with inclusions derived from Clone 9 are eliminated more rapidly from the population. Error bars = S.E.M, **** = p<0.0001.
(I) Clone 9-transduced cells grow more slowly. After transduction of stable cells, colonies with inclusions derived from Clone 9 have fewer cells than colonies with inclusions derived from Clone 10. Colonies without inclusions have identical cell numbers (error bars = S.E.M, **** = p<0.0001). See for differences in cell growth rate in tau RD(LM)-HA cells and for LDH toxicity assay in tau RD(LM)-HA background.
(J) Clones 1, 9, and 10 maintain distinctive morphologies after 6 months in culture. See also for data indicating that juxtanuclear Clone 10 but not Clone 9 inclusions are aggresomes.
(K) Structural characteristics (limited proteolysis digestion patterns) of strains are propagated with high fidelity over six months.