Fig. 4.

Increased fusion protein expression improves assay sensitivity for 4R tau prions. HEK293T cells with a higher expression of the same fusion protein as the Tau(4RD*LM)–YFP(1) cells were created [Tau(4RD*LM)–YFP(2) cells]. (A) Western blot analysis of Tau(4RD*LM)–YFP(1) and Tau(4RD*LM)–YFP(2) cells in the absence and presence of infection with lysate from clone 9 cells, which stably propagate synthetic tau prions. The 4RD*LM–YFP construct was probed with anti-GFP (green fluorescent protein) antibody (Top). The membrane was reprobed for vinculin as a loading control (Bottom). (B) Quantification of protein expression in the Western blot was performed using ImageJ software (NIH). Expression levels were normalized to basal 4RD*LM–YFP expression in HEK293 cells. Data shown as mean ± SD (n = 2). (C and D) Crude brain homogenates from control (n = 6), PiD (n = 6), AGD (n = 2), CBD (n = 5), and PSP (n = 6) patient samples were diluted in DPBS and incubated with Tau(4RD*LM)–YFP(2) cells for 4 d. (C) Quantification of cell infection after incubation was determined by dividing the total fluorescence in each image by the total cell count. Tau prions from the 4R tauopathies were all capable of infecting the Tau(4RD*LM)–YFP(2) cells (AGD, P < 0.001; CBD, P < 0.05; PSP, P < 0.001), whereas the control samples did not. Of the six PiD samples, two infected the Tau(4RD*LM)–YFP(2) cells (PiD3 and PiD4) and the other four did not (P = 0.41). *P < 0.05. Data are shown as the mean of five images per well in six wells. All values are shown in Table S2. (D) Representative images of HEK293T cells infected with AGD, CBD, and PSP but not control or PiD patient samples. YFP is shown in green. (Scale bar, 50 μm.)