Fig. 2: AS-141G PFFs induce formation of PLA signal in human neurons but added PFFs themselves are undetected.

a Human cortical neurons were cultured for 35 days before treatment with either S129A PFFs, AS-141G PFFs (S129A-α-synuclein-141G), or PBS, and fixed after 2 h or 7 days. b Representative images from 2 h post treatment cultures immunostained with MJF-14 PLA (red), pS129-α-synuclein (grey), βIII-tubulin/TUJ1 (purple), and DAPI nuclear stain (blue). Arrows indicate examples of PLA signals. Scale bar = 20 µm. c Quantification of the number of PLA particles per cell after 2 h, as determined by the MJF-14 PLA. Treatment significantly affects PLA signal density (F(2,269) = [47.79], p < 0.0001), with the S129A PFF group differing from both PBS and AS-141G PFF groups (p < 0.0001) while PBS and AS-141G PFF groups do no differ significantly (p = 0.2318). d Representative images from 7 days post treatment cultures immunostained with MJF-14 PLA (red), pS129-α-synuclein (grey), βIII-tubulin (purple), and DAPI nuclear stain (blue). Arrows indicate examples of PLA signals. Scale bar = 20 µm. e Quantification of the number of PLA particles per cell after 7 days, as determined by the MJF-14 PLA. Treatment significantly affects PLA signal density (F(2,296) = [35.26], p < 0.0001), with the PBS group differing from both S129A and AS-141G PFF groups (p < 0.0001) while S129A and AS-141G PFF groups do no differ significantly (p = 0.5009). f Magnified panels from (d) with arrows indicating the co-detection of particles with both MJF-14 PLA (red) and pS129-α-synuclein (grey). Scale bar = 5 µm. Graphs display mean ± SEM from two independent replicates and each dot signifies one image. Experiments were performed two times independently with two technical replicates each time, and groups were compared using a two-way ANOVA followed by Tukey’s multiple comparison test. ****p < 0.0001.