Fig. 1: MJF-14 PLA signal is directly dependent upon aggregate formation in α-synuclein transgenic neuroblastoma cells.

a Principle of the proximity ligation assay for detection of α-synuclein aggregates. b Experimental setup: α-synuclein transgenic SH-SY5Y cells are differentiated into non-mitotic neuronal-like cells using retinoic acid, treated ± doxycycline to mediate α-synuclein overexpression and ± ASI1D to modulate α-synuclein aggregation. Then, cells are fixed and subjected to PLA using the α-synuclein conformation-specific MJFR-14-6-4-2 antibody or the total α-synuclein syn211 antibody. c Representative images of MJF-14 PLA in conditions without α-synuclein expression, with α-synuclein expression, and with α-synuclein expression + ASI1D-treatment. PLA signal is displayed in red (small dots), α-tubulin in grey and DAPI in blue. Arrows indicate examples of PLA particles in low abundance conditions. Scale bar = 20 µm. d Quantification of the number of PLA particles per cell, as determined by the MJF-14 PLA. The +α-syn condition is significantly different from both -α-syn and +α-syn + ASI1D (p < 0.0001). e Representative images of syn211 PLA in conditions without α-synuclein expression, with α-synuclein expression, and with α-synuclein expression + ASI1D-treatment. PLA signal is displayed in red (small dots), α-tubulin in grey and DAPI in blue. Arrows indicate examples of PLA particles in low abundance conditions. Scale bar = 20 µm. f Quantification of the number of PLA particles per cell, as determined by the syn211 PLA. The +α-syn condition is significantly different from -α-syn (p < 0.0001) but not +α-syn + ASI1D (p > 0.9999). Graphs display mean ± SEM from one replicate and each dot signifies one image. Experiments were performed minimum three times independently, and groups were compared using a Kruskal-Wallis one-way ANOVA followed by the Dunn post hoc test. ****p < 0.0001.