( E ) . Co - immunoprecipitation of exogenous biotin - labelled Fib - Tau with α3 - NKA , GluA2 GluN1 . α3 - NKA , GluA2 GluN1 were immunoprecipitated using specific antibodies as described in methods section . presence of Fib - Tau in immunoprecipitate was assessed using a slot blot apparatus nitrocellulose membranes probed with streptavidin - HRP . A 2 . 4 - , 2 . 3 - , 1 . 8 - fold enrichment in Tau band - is observed in α3 - NKA , GluA2 GluN1 immunoprecipitates , respectively , compared to controls performed with pre - immune goat or rabbit IgGs . Co - immunoprecipitates of exogenous biotin - labelled Fib - Tau with anti α3 - NKA , GluA2 GluN1 specific antibodies were also subjected to SDS - PAGE Western blot . presence of Fib - Tau in immunoprecipitates was assessed by probing nitrocellulose membranes with streptavidin - HRP . Fib - Tau co - immunoprecipitates with α3 - NKA GluA2 - AMPA receptor but not with GluN1 - NMDA receptor .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O B-EXP_ASSAY O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O O O O B-ORGANISM O B-ORGANISM O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O B-GENEPROD O B-SMALL_MOLECULE O O

(E). Co-immunoprecipitation of exogenous biotin-labelled Fib-Tau with α3-NKA, GluA2 and GluN1. α3-NKA, GluA2 and GluN1 were immunoprecipitated using specific antibodies as described in the methods section. The presence of Fib-Tau in the immunoprecipitate was assessed using a slot blot apparatus and nitrocellulose membranes probed with streptavidin-HRP. A 2.4-, 2.3-, and 1.8- fold enrichment in Tau band-intensity is observed in α3-NKA, GluA2 and GluN1 immunoprecipitates, respectively, compared to controls performed with pre-immune goat or rabbit IgGs. Co-immunoprecipitates of exogenous biotin-labelled Fib-Tau with anti α3-NKA, GluA2 and GluN1 specific antibodies were also subjected to SDS-PAGE and Western blot analysis. The presence of Fib-Tau in the immunoprecipitates was assessed by probing the nitrocellulose membranes with streptavidin-HRP. Fib-Tau co-immunoprecipitates with α3-NKA and GluA2-AMPA receptor but not with GluN1-NMDA receptor.


( A , B ) neurons were exposed to Fib - Tau - ATTO550 ( 0 . 36nM , red ) for 10 ( top row ) or 60 min ( bottom row ) excitatory ( Homer , green ) inhibitory synapses ( Gephyrin , blue ) were immuno - labelled . Fib - Tau clusters co - localized / or apposed to Homer ( arrowheads ) but not Gephyrin ( A ) . Quantitative shows that 10 to 20 % of Fib - Tau clusters are localized at Homer containing synapses ( B ) . Box - plot represents : median , interquartile range 10 - 90 % distribution , Unpaired t - to compare difference between 10 60 min , n is number of analyzed from 3 - experiments ( 10 min : 49 ; 60 min : 50 ) . Scale bar , 5µm .

O O O O O B-CELL_TYPE O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-GENEPROD O O O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-EXP_ASSAY O B-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A, B) Primary neurons were exposed to Fib-Tau-ATTO550 (0.36nM, red) for 10 (top row) or 60 min (bottom row) and excitatory (Homer, green) and inhibitory synapses (Gephyrin, blue) were immuno-labelled. Fib-Tau clusters co-localized and/or apposed to Homer (arrowheads) but not Gephyrin (A). Quantitative analysis shows that 10 to 20% of Fib-Tau clusters are localized at Homer containing synapses (B). Box-plot represents: median, interquartile range and 10-90% distribution, Unpaired t-test to compare difference between 10 and 60 min, n is number of images analyzed from 3-experiments (10 min: 49; 60 min: 50). Scale bar, 5µm.


Exposure of neurons to Fib - Tau - ATTO550 ( 0 . 36nM , 60 min , red ) immuno - labelling of excitatory synapse ( Anti - Rabbit - Homer or Anti - Mouse - PSD , blue ) α3 - NKA or GluA1 - AMPA or GluA2 - AMPA or GluN1 - NMDA or GluN2B - NMDA subunits ( green , post - permeabilization ) . Arrows indicate excitatory synapses where Fib - Tau α3 - NKA / AMPA / NMDA are co - localized ( C ) . Mann - Whitney - , n is number of analyzed from 3 - 4 experiments ( α3 - NKA : 75 ; GluA1 : 95 ; GluA2 : 50 ; GluN1 / N2B : 45 ) .

O O B-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR O O O B-ORGANISM O B-GENEPROD O O O B-ORGANISM O B-SUBCELLULAR O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O B-GENEPROD O B-SMALL_MOLECULE O B-GENEPROD O B-SMALL_MOLECULE O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O

Exposure of neurons to Fib-Tau-ATTO550 (0.36nM, 60 min, red) and immuno-labelling of excitatory synapse (Anti-Rabbit-Homer or Anti-Mouse-PSD, blue) and α3-NKA or GluA1-AMPA or GluA2-AMPA or GluN1-NMDA or GluN2B-NMDA subunits (green, post-permeabilization). Arrows indicate excitatory synapses where Fib-Tau and α3-NKA/AMPA/NMDA are co-localized (C). Mann-Whitney-test, n is number of images analyzed from 3-4 experiments (α3-NKA: 75; GluA1: 95; GluA2: 50; GluN1/N2B: 45).


Exposure of neurons to Fib - Tau - ATTO550 ( 0 . 36nM , 60 min , red ) immuno - labelling of excitatory synapse ( Anti - Rabbit - Homer or Anti - Mouse - PSD , blue ) α3 - NKA or GluA1 - AMPA or GluA2 - AMPA or GluN1 - NMDA or GluN2B - NMDA subunits ( green , post - permeabilization ) . of ( indicating of clusters of synaptic α3 - NKA / AMPA / NMDA spots ( obtained after thresholding ) following exposure to Fib - Tau showed a reduction in of α3 - NKA increase in of GluA2 - subunit containing AMPA receptors ( D ) . Box - plot represents : median , interquartile range 10 - 90 % distribution , * * p < 0 . 01 ; ns = not significant . Scale bar , 5µm .

O O B-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR O O O B-ORGANISM O B-GENEPROD O O O B-ORGANISM O B-SUBCELLULAR O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O B-GENEPROD O B-SMALL_MOLECULE O B-GENEPROD O B-SMALL_MOLECULE O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Exposure of neurons to Fib-Tau-ATTO550 (0.36nM, 60 min, red) and immuno-labelling of excitatory synapse (Anti-Rabbit-Homer or Anti-Mouse-PSD, blue) and α3-NKA or GluA1-AMPA or GluA2-AMPA or GluN1-NMDA or GluN2B-NMDA subunits (green, post-permeabilization). Quantification of the fluorescence intensity (indicating size of clusters of synaptic α3-NKA/AMPA/NMDA spots (obtained after thresholding) following exposure to Fib-Tau showed a reduction in the size of α3-NKA and increase in the size of GluA2-subunit containing AMPA receptors (D). Box-plot represents: median, interquartile range and 10-90% distribution, **p<0.01; ns= not significant. Scale bar, 5µm.


( A - C ) SPT - QD of pHluorin tagged AMPA ( GluA1 GluA2 ) NMDA ( GluN1 GluN2B ) receptor subunits using GFP - antibody pre - coupled to QD655 . neurons were exposed or not to Fib - Tau ( 0 . 36nM , 60 min ) . Cumulative frequency of diffusion coefficient of trajectories ( extracted from MSD plot , inset , A ) show a slow - down in diffusion of GluA2 - subunit but not GluA1 , GluN1 GluN2B subunits ( A ) . To compute statistical differences , diffusion coefficient of trajectories per movie ( from 3 independent experiments ) were averaged ( B ) . To measure statistical differences in MSD , explored ( under curve during 600 - 900ms of MSD plot ) per movie ( from 3 independent experiments ) were averaged ( C ) . Decreased diffusion coefficient explored for GluA2 - AMPA was observed indicating increased confinement concomitant with increased clustering ( Fig 5C ) following exposure to Fib - Tau . Mann - Whitney - , n is number of movies ( left to right ) : 14 , 15 , 14 , 13 , 15 , 15 , 15 , 15 . * * p < 0 . 01 ; ns = not significant .

O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O B-CELL_TYPE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A-C) SPT-QD of pHluorin tagged AMPA (GluA1 and GluA2) and NMDA (GluN1 and GluN2B) receptor subunits using GFP-antibody pre-coupled to QD655. Primary neurons were exposed or not to Fib-Tau (0.36nM, 60 min). Cumulative frequency of diffusion coefficient of all trajectories (extracted from MSD plot, inset, A) show a slow-down in diffusion of GluA2-subunit but not GluA1, GluN1 and GluN2B subunits (A). To compute statistical differences, the diffusion coefficient of all trajectories per movie (from 3 independent experiments) were averaged (B). To measure statistical differences in MSD, the explored area (area under curve during 600-900ms of MSD plot) per movie (from 3 independent experiments) were averaged (C). Decreased diffusion coefficient and area explored for GluA2-AMPA was observed indicating increased confinement concomitant with increased clustering (Fig 5C) following exposure to Fib-Tau. Mann-Whitney-test, n is number of movies (left to right): 14, 15, 14, 13, 15, 15, 15, and 15. **p<0.01; ns= not significant.


( A ) Single Particle Tracking using PALM ( SPT - PALM ) was performed on α3 - NKA - Dendra expressing neurons . A representative image shows an epifluorescence image trajectories ( strands of different colors ) of minimum 5 points in . Scale bars : 1 µm in panel ( A )

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O B-EXP_ASSAY O B-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O

(A) Single Particle Tracking using PALM (SPT-PALM) was performed on α3-NKA-Dendra expressing primary neurons. A representative image shows an epifluorescence image and trajectories (strands of different colors) of minimum 5 points in length. Scale bars: 1 µm in panel (A)


( B , C ) Extraction of diffusion coefficient using mean squared displacement ( MSD ) approach using minimum 5 - point trajectory . Spine shaft were visually identified . An upward shift of MSD curve , indicating reduced confinement was observed following exposure to Fib - Tau ( 0 . 36nM , 60 min ) ( B ) . As expected , an increased diffusion coefficient ( i . e . increased mobility ) ( C ) of α3 - NKA is observed in dendritic shaft following exposure to Fib - Tau . Mann - Whitney - , n = 21 , obtained from 4 independent experiments ; Each dot represents average diffusion coefficient of trajectories value per . Data information : * p < 0 . 05 ; ns = not significant .

O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O

(B, C) Extraction of diffusion coefficient using mean squared displacement (MSD) approach using minimum 5-point trajectory length. Spine and shaft were visually identified. An upward shift of MSD curve, indicating reduced confinement was observed following exposure to Fib-Tau (0.36nM, 60 min) (B). As expected, an increased diffusion coefficient (i.e. increased mobility) (C) of α3-NKA is observed in dendritic shaft following exposure to Fib-Tau. Mann-Whitney-test, n=21 cells, obtained from 4 independent experiments; Each dot represents average diffusion coefficient of all trajectories value per cell. Data information: *p<0.05; ns= not significant.


Extraction of number of diffusive states of α3 - NKA transition rates between these diffusive states using thousands of short ( minimum 2 - point ) trajectories using Hidden Markov Model occupancy time of α3 - NKA in bound - state also decreased whereas that in free - state increased upon neuron exposure to Fib - Tau ( E ) . Box - plot represents : median , interquartile range entire distribution , Mann - Whitney - on averaged value per movie from 4 experiments ( n = 21 ) . Data information : * p < 0 . 05 ; ns = not significant .

O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-CELL_TYPE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Extraction of the number of diffusive states of α3-NKA and the transition rates between these diffusive states using thousands of short (minimum 2-point) trajectories using Hidden Markov Model The occupancy time of α3-NKA in bound-state also decreased whereas that in free-state increased upon neuron exposure to Fib-Tau (E). Box-plot represents: median, interquartile range and entire distribution, Mann-Whitney-test on averaged value per movie from 4 experiments (n=21 cells). Data information: *p<0.05; ns= not significant.


Extraction of number of diffusive states of α3 - NKA transition rates between these diffusive states using thousands of short ( minimum 2 - point ) trajectories using Hidden Markov Model Dwell - time of α3 - NKA is also reduced in bound state indicating destabilization of native α3 - NKA - complexes following interaction with Fib - Tau ( F ) . Box - plot represents : median , interquartile range entire distribution , Mann - Whitney - on averaged value per movie from 4 experiments ( n = 21 ) . Data information : * p < 0 . 05 ; ns = not significant .

O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Extraction of the number of diffusive states of α3-NKA and the transition rates between these diffusive states using thousands of short (minimum 2-point) trajectories using Hidden Markov Model Dwell-time of α3-NKA is also reduced in bound state indicating destabilization of native α3-NKA-complexes following interaction with Fib-Tau (F). Box-plot represents: median, interquartile range and entire distribution, Mann-Whitney-test on averaged value per movie from 4 experiments (n=21 cells). Data information: *p<0.05; ns= not significant.


( G - H ) - surface biotinylation on neurons exposed or not to Fib - Tau ( 1 . 8nM , 60 min ) . amount of α - NKA at surface decreased upon exposure of neurons to Fib - Tau ( G ) . was performed by normalizing western blot band with input . Paired - t - , 3 independent experiments . Data information : * p < 0 . 05 ; ns = not significant .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-SUBCELLULAR O O O O B-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G-H) Cell-surface biotinylation on primary neurons exposed or not to Fib-Tau (1.8nM, 60 min). The amount of α-NKA at cell surface decreased upon exposure of neurons to Fib-Tau (G). Quantification was performed by normalizing the western blot band intensity with input. Paired-t-test, 3 independent experiments. Data information: *p<0.05; ns= not significant.


( I ) Paraformaldehyde fixation α3 - NKA - pHluorin ( green , top row ) reveals endocytic spots otherwise not visible in live neurons ( green , bottom row ) . Many Fib - Tau - ATTO647 ( red ) clusters overlapped ( arrow ) with α3 - NKA - pHluorin spots ( exposure , 0 . 36nM , 60 min ) . Scale bars

O O O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O

(I) Paraformaldehyde fixation α3-NKA-pHluorin (green, top row) reveals endocytic spots otherwise not visible in live neurons (green, bottom row). Many Fib-Tau-ATTO647 (red) clusters overlapped (arrow) with α3-NKA-pHluorin spots (exposure, 0.36nM, 60 min). Scale bars 2µm


( J ) majority of overlapping Fib - Tau - ATTO647 clusters ( red ) α3 - NKA - pHluorin spots ( green ) localized on top of Clathrin - mRFP ( blue ) . Scale bars :

O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE B-SUBCELLULAR O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O O O O O O O

(J) The majority of the overlapping Fib-Tau-ATTO647 clusters (red) and α3-NKA-pHluorin spots (green) localized on top of Clathrin-mRFP (blue) puncta. Scale bars: 2µm


DIV21 neurons were co - exposed to fixed of Fib - Tau ( 0 . 36nM , 60min , ATTO550 labelled , red ) increasing concentrations ( given in monomer - equivalent ) of α - Syn fibrils [ in total 5 - experiments were performed with either ATTO647 labeled α - Syn ( shown here in green ) or unlabeled α - Syn A - dependent increase in binding clustering of α - Syn fibrils is seen . Notably , Fib - Tau clustering dramatically increased with increasing concentrations of α -

O B-CELL_TYPE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD

DIV21 neurons were co-exposed to fixed concentration of Fib-Tau (0.36nM, 60min, ATTO550 labelled, red) and increasing concentrations (given in monomer-equivalent) of α-Syn fibrils [in total 5-experiments were performed with either ATTO647 labeled α-Syn (shown here in green) or unlabeled α-Syn A concentration-dependent increase in the binding and clustering of α-Syn fibrils is seen. Notably, Fib-Tau clustering dramatically increased with increasing concentrations of α-Syn


DIV21 neurons were co - exposed to fixed of Fib - Tau ( 0 . 36nM , 60min , ATTO550 labelled , red ) increasing Aβ ( given in monomer - equivalent ) of Aβ ( B ) fibrils [ in total 5 - experiments were performed with either ATTO647 labeled Aβ ( shown here in green ) or unlabeled Aβ fibrils ] . - dependent increase in binding clustering of Aβ fibrils is seen . Notably , Fib - Tau clustering dramatically increased with increasing concentrations of α - Syn , but not Aβ

O B-CELL_TYPE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-SMALL_MOLECULE

DIV21 neurons were co-exposed to fixed concentration of Fib-Tau (0.36nM, 60min, ATTO550 labelled, red) and increasing Aβ (given in monomer-equivalent) of Aβ (B) fibrils [in total 5-experiments were performed with either ATTO647 labeled Aβ (shown here in green) or unlabeled Aβ fibrils]. concentration-dependent increase in the binding and clustering of Aβ fibrils is seen. Notably, Fib-Tau clustering dramatically increased with increasing concentrations of α-Syn, but not Aβ fibrils


DIV21 neurons were co - exposed to fixed of Fib - Tau ( 0 . 36nM , 60min , ATTO550 labelled , red ) increasing concentrations ( given in monomer - equivalent ) of α - Syn fibrils [ in total 5 - experiments were performed with either ATTO647 labeled α - Syn or unlabeled α - Syn A - dependent increase in binding clustering of both α - Syn fibrils is seen . One - Way ANOVA with Dunnett ' s post - hoc was performed to compare different condition to reference condition ( 100nM ) . number of analyzed is 27 , 25 , 25 , 26 28 in ( C ) , from left to right . data originate from 3 to 4 independent experiments . Data information : Box - plot represents : median , interquartile range 10 - 90 % distribution , Mann - Whitney - is performed , n = 40 from 4 - experiments , Scale bars , 5µm . * p < 0 . 05 , * * * p < 0 . 001 ; ns = not significant .

O B-CELL_TYPE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

DIV21 neurons were co-exposed to fixed concentration of Fib-Tau (0.36nM, 60min, ATTO550 labelled, red) and increasing concentrations (given in monomer-equivalent) of α-Syn fibrils [in total 5-experiments were performed with either ATTO647 labeled α-Syn or unlabeled α-Syn A concentration-dependent increase in the binding and clustering of both α-Syn fibrils is seen. One-Way ANOVA with Dunnett's post-hoc test was performed to compare the different condition to the reference condition (100nM).The number of images analyzed is 27, 25, 25, 26 and 28 in (C) , from left to right. The data originate from 3 to 4 independent experiments. Data information: Box-plot represents: median, interquartile range and 10-90% distribution, Mann-Whitney-test is performed, n=40 images from 4-experiments, Scale bars, 5µm. *p<0.05, ***p<0.001; ns= not significant.


DIV21 neurons were co - exposed to fixed of Fib - Tau ( 0 . 36nM , 60min , ATTO550 labelled , red ) increasing concentrations ( given in monomer - equivalent ) of Aβ fibrils [ in total 5 - experiments were performed with either ATTO647 labeled Aβ or unlabeled Aβ fibrils A - dependent increase in binding clustering of Aβ fibrils is seen . One - Way ANOVA with Dunnett ' s post - hoc was performed to compare different condition to reference condition ( 100nM ) . number of analyzed is 18 , 50 , 50 , 50 , 25 in ( D ) , from left to right . data originate from 3 to 4 independent experiments . Data information : Box - plot represents : median , interquartile range 10 - 90 % distribution , Mann - Whitney - is performed , n = 40 from 4 - experiments , Scale bars , 5µm . * p < 0 . 05 , * * * p < 0 . 001 ; ns = not significant .

O B-CELL_TYPE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

DIV21 neurons were co-exposed to fixed concentration of Fib-Tau (0.36nM, 60min, ATTO550 labelled, red) and increasing concentrations (given in monomer-equivalent) of Aβ fibrils [in total 5-experiments were performed with either ATTO647 labeled Aβ or unlabeled Aβ fibrils A concentration-dependent increase in the binding and clustering of Aβ fibrils is seen. One-Way ANOVA with Dunnett's post-hoc test was performed to compare the different condition to the reference condition (100nM).The number of images analyzed is 18, 50, 50, 50, 25 in (D), from left to right. The data originate from 3 to 4 independent experiments. Data information: Box-plot represents: median, interquartile range and 10-90% distribution, Mann-Whitney-test is performed, n=40 images from 4-experiments, Scale bars, 5µm. *p<0.05, ***p<0.001; ns= not significant.


Notably , Fib - Tau clustering dramatically increased with increasing concentrations of α - Syn , but not Aβ fibrils in ( E ) includes 5 - experiments ( n = number of analyzed , left to right : 58 , 54 , 50 , 62 , 63 , 65 , 43 , 35 , 60 , 60 , 60 , 35 ; One - Way ANOVA with Dunnette ' s post - hoc was performed to compare of Fib - Tau clusters ) . Data information : Box - plot represents : median , interquartile range 10 - 90 % distribution , Mann - Whitney - is performed , n = 40 from 4 - experiments , Scale bars , 5µm . * p < 0 . 05 , * * * p < 0 . 001 ; ns = not significant .

O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Notably, Fib-Tau clustering dramatically increased with increasing concentrations of α-Syn, but not Aβ fibrils The quantification in (E) includes all 5-experiments (n = number of images analyzed, left to right: 58, 54, 50, 62, 63, 65, 43, 35, 60, 60, 60, 35; One-Way ANOVA with Dunnette's post-hoc test was performed to compare the size of Fib-Tau clusters). Data information: Box-plot represents: median, interquartile range and 10-90% distribution, Mann-Whitney-test is performed, n=40 images from 4-experiments, Scale bars, 5µm. *p<0.05, ***p<0.001; ns= not significant.


A perfect co - localization between Fib - Tau α - Syn clusters is also observed . proportion of Fib - Tau clusters co - localizing with α - Syn fibrils increased with an increasing of α - Syn fibrils reaching 60 - 70 % overlap A weaker , independent , overlap ( 10 - 20 % ) was observed with Aβ fibrils in ( F ) is derived from 3 to 4 independent experiments . number of analyzed is 27 , 25 , 25 , 26 , 28 for α - Syn 18 , 50 , 50 , 50 , 25 for Aβ from left to right . One - Way ANOVA with Dunnett ' s post - hoc was performed to compare co - localization differences from 100nM . Box - plot represents : median , interquartile range 10 - 90 % distribution . Data information : Box - plot represents : median , interquartile range 10 - 90 % distribution , Mann - Whitney - is performed , n = 40 from 4 - experiments , Scale bars , 5µm . * p < 0 . 05 , * * * p < 0 . 001 ; ns = not significant .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD B-SUBCELLULAR O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A perfect co-localization between Fib-Tau and α-Syn clusters is also observed. The proportion of Fib-Tau clusters co-localizing with α-Syn fibrils increased with an increasing concentration of α-Syn fibrils reaching 60-70% overlap A weaker, concentration independent, overlap (10-20%) was observed with Aβ fibrils The quantification in (F) is derived from 3 to 4 independent experiments. The number of images analyzed is 27, 25, 25, 26, 28 for α-Syn and 18, 50, 50, 50, 25 for Aβ from left to right. One-Way ANOVA with Dunnett's post-hoc test was performed to compare the co-localization differences from 100nM concentration. Box-plot represents: median, interquartile range and 10-90% distribution. Data information: Box-plot represents: median, interquartile range and 10-90% distribution, Mann-Whitney-test is performed, n=40 images from 4-experiments, Scale bars, 5µm. *p<0.05, ***p<0.001; ns= not significant.


( G - I ) neurons were co - exposed to Fib - Tau ( 0 . 36nM , ATTO550 labelled ) Fib - α - Syn ( 50nM , monomer - equivalent , unlabeled ) for 60min immunolabelled for homer ( G ) or α3 - NKA ( H ) . of Fib - Tau clusters co - localized with homer or α3 - NKA ( I ) . Note an increased co - localization of Fib - Tau with homer in presence of Fib - α - Syn . Data information : Box - plot represents : median , interquartile range 10 - 90 % distribution , Mann - Whitney - is performed , n = 40 from 4 - experiments , Scale bars , 5µm . * p < 0 . 05 , * * * p < 0 . 001 ; ns = not significant .

O O O O O B-CELL_TYPE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G-I) Primary neurons were co-exposed to Fib-Tau (0.36nM, ATTO550 labelled) and Fib-α-Syn (50nM, monomer-equivalent, unlabeled) for 60min and immunolabelled for homer (G) or α3-NKA (H). Quantification of Fib-Tau clusters co-localized with homer or α3-NKA (I). Note an increased co-localization of Fib-Tau with homer in presence of Fib-α-Syn. Data information: Box-plot represents: median, interquartile range and 10-90% distribution, Mann-Whitney-test is performed, n=40 images from 4-experiments, Scale bars, 5µm. *p<0.05, ***p<0.001; ns= not significant.


( A ) A boxplot showing log2 TPM values of HSPA5 gene expression compared between tumors matched normal samples across 23 types from TCGA with normal tissue data available . boxplot indicates median ( central band ) interquartile range ( boxes ) of expression value whiskers indicates values that outside of middle 50 % ( interquartile ) . barplot on top shows number of normal tissue samples for each corresponding type . Asterisks indicate significant differences by student ' s t - after Benjamini - Hochberg multiple testing correction ( FDR < 0 . 05 ) .

O O O O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) A boxplot showing log2 TPM values of HSPA5 gene expression compared between tumors and matched normal samples across 23 tumor types from TCGA with normal tissue data available. The boxplot indicates the median (central band) and the interquartile range (boxes) of the expression value and whiskers indicates values that outside of the middle 50% (interquartile). The barplot on the top shows the number of normal tissue samples for each corresponding tumor type. Asterisks indicate significant differences by student's t-test after Benjamini-Hochberg multiple testing correction (FDR < 0.05).


( B ) of Xbp1 splicing in DLD1 SKOV3 treated with varying concentrations of Reversine ( Rv ) for 12 , 24 , 48 , 72 hours . Thapsigargin ( Tg ) is shown as positive control .

O O O O B-GENEPROD B-EXP_ASSAY O B-CELL_LINE I-CELL_LINE O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O

(B) Analysis and quantification of Xbp1 splicing in DLD1 and SKOV3 cells treated with varying concentrations of Reversine (Rv) for 12, 24, 48, and 72 hours. Thapsigargin (Tg) is shown as positive control.


( D ) Xbp1 splicing in B16 melanoma eight B16 : MEF fused clonal lines .

O O O B-GENEPROD B-EXP_ASSAY O B-CELL_LINE I-CELL_LINE O B-CELL_LINE O B-CELL_TYPE O O O O

(D) Xbp1 splicing in B16 melanoma cells and eight B16:MEF fused clonal cell lines.


( E ) Detection of mRNA expression ( by RT - PCR ) of UPR - associated genes ( Ern1 , Af4 , Ddit3 Atf6 ) in treated with Reversine ( Rv ) at 4µM ( DLD1 ) or 0 . 5µM ( SKOV3 ) for 6 hours . Data points refer to triplicate samples collected at same time point , run in duplicate , expressed as means ± SD .

O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-CELL_LINE O O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Detection of mRNA expression (by RT-PCR) of UPR-associated genes (Ern1, Af4, Ddit3 and Atf6) in tumor cells treated with Reversine (Rv) at 4µM (DLD1 cells) or 0.5µM (SKOV3 cells) for 6 hours. Data points refer to triplicate samples collected at the same time point, run in duplicate, and expressed as means ± SD.


( F ) Western Blot of activation of IRE1 , PERK ATF branches of UPR in DLD1 SKOV3 treated or not with Rv at 4µM for 24H ( DLD1 ) or 0 . 5µM for 3 days ( SKOV3 ) . Thapsigargin ( Tg ) was used as positive control .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O B-CELL_LINE I-CELL_LINE O O O O B-SMALL_MOLECULE O O O O O B-CELL_LINE O O O O O O O O O B-CELL_LINE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O

(F) Western Blot analysis of the activation of IRE1, PERK and ATF branches of the UPR in DLD1 and SKOV3 cells treated or not with Rv at 4µM for 24H (DLD1 cells) or 0.5µM for 3 days (SKOV3 cells). Thapsigargin (Tg) was used as positive control.


( G ) Western Blot of phosphorylation of GCN2 PKR defining integrated stress response ( ISR ) in DLD1 SKOV3 treated or not with Rv as in F ) . starved for 24 hours then treated for 30 min with Calyculin - A ( Cal - A ) were used as positive control .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O O O O O B-CELL_LINE I-CELL_LINE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O

(G) Western Blot analysis of phosphorylation of GCN2 and PKR defining the integrated stress response (ISR) in DLD1 and SKOV3 cells treated or not with Rv as in F). Cells starved for 24 hours then treated for 30 min with Calyculin-A (Cal-A) were used as positive control.


( A ) Xbp1 splicing ( left panel ) ( right panel ) of bone marrow - derived macrophages ( BMDM ) cultured in conditioned medium ( CM ) of Rv - treated DLD1 SKOV3 or respective control medium ( culture medium of cancer not treated with Rv ) , CM of fused B16 melanoma or their nonfused parental .

O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O O O O O O O O B-SMALL_MOLECULE O O B-CELL_LINE I-CELL_LINE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-CELL_LINE I-CELL_LINE O O O O O

(A) Xbp1 splicing analysis (left panel) and quantification (right panel) of bone marrow-derived macrophages (BMDM) cultured in conditioned medium (CM) of Rv-treated DLD1 and SKOV3 cells or respective control medium (culture medium of cancer cells not treated with Rv), and CM of fused B16 melanoma cells or their nonfused parental cells.


( B , C ) mRNA expression ( RT - PCR ) of Il6 ( B ) Arg1 ( C ) in BMDM cultured in CM of Rv - treated SKOV3 ( 0 . 5µM for 3 days ) or control , CM of fused B16 or their nonfused parental . Data points refer to triplicate experiments expressed as means ± SD .

O O O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O B-GENEPROD O O O O B-CELL_TYPE O O O O B-SMALL_MOLECULE O O B-CELL_LINE O O O O O O O O O O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O

(B, C) mRNA expression (RT-PCR) of Il6 (B) and Arg1 (C) in BMDM cultured in the CM of Rv-treated SKOV3 cells (0.5µM for 3 days) or control cells, and CM of fused B16 cells or their nonfused parental cells. Data points refer to triplicate experiments expressed as means ± SD.


( E ) Flow cytometry of IFNγ Granzyme B production by human T activated by anti - CD3 / anti - CD28 Dynabeads in presence of CM from Rv - treated SKOV3 or control SKOV3 ( cultured without Rv ) . Column graphs represent expression of IFNγ ( upper panel ) Granzyme B ( lower panel ) . Percentages in parenthesis refer to change from control . Data representative triplicate samples at same time point repeated at least twice . Data are expressed as means ± SD .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O B-ORGANISM B-CELL_TYPE O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O B-CELL_LINE O O B-CELL_LINE O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Flow cytometry analysis of IFNγ and Granzyme B production by human T cells activated by anti-CD3/anti-CD28 Dynabeads in the presence of CM from Rv-treated SKOV3 cells or control SKOV3 cells (cultured without Rv). Column graphs represent the percentage expression of IFNγ (upper panel) and Granzyme B (lower panel). Percentages in parenthesis refer to change from control. Data representative triplicate samples at the same time point repeated at least twice. Data are expressed as means ± SD.


( F ) Flow cytometry of IFNγ Granzyme B production by human T activated by anti - CD3 / anti - CD28 Dynabeads in complete medium spiked with Rv ( 0 . 5µM ) . Column graphs represent expression of IFNγ ( upper panel ) Granzyme B ( lower panel ) . Data representative triplicate samples at same time point repeated at least twice . Data are expressed as means ± SD .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O B-ORGANISM B-CELL_TYPE O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Flow cytometry analysis of IFNγ and Granzyme B production by human T cells activated by anti-CD3/anti-CD28 Dynabeads in complete medium spiked with Rv (0.5µM). Column graphs represent the percentage expression of IFNγ (upper panel) and Granzyme B (lower panel). Data representative triplicate samples at the same time point repeated at least twice. Data are expressed as means ± SD.


( G ) IFNγ ( left ) Granzyme B ( right ) production by human T activated by anti - CD3 / anti - CD28 Dynabeads cultured in CM from fused B16 melanoma or their parental non - fused . Percentages in parenthesis refer to change from parental non - fused . Data representative triplicate samples at same time point repeated at least twice . Data are expressed as means ± SD .

O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD O O O O O B-ORGANISM B-CELL_TYPE O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) IFNγ (left) and Granzyme B (right) production by human T cells activated by anti-CD3/anti-CD28 Dynabeads cultured in CM from fused B16 melanoma cells or their parental non-fused cells. Percentages in parenthesis refer to change from parental non-fused cells. Data representative triplicate samples at the same time point repeated at least twice. Data are expressed as means ± SD.


A Kinetics of intracellular Ca2 + after EDN stimulation . UACC257 A2058 ( WT ENDRB - KO ) were seeded on 96 well plates , loaded with Fluo - 4 dye stimulated with 100 nM EDN or PBS . Fluo - 4 was monitored over ten minutes ( n = 6 ) . Representative example of three independent experiments .

O B-EXP_ASSAY O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD O O B-CELL_LINE I-CELL_LINE O O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O

A Kinetics of intracellular Ca2+ after EDN stimulation. UACC257 and A2058 (WT and ENDRB-KO) were seeded on 96 well plates, loaded with Fluo-4 dye and stimulated with 100 nM EDN or PBS. Fluo-4 fluorescence was monitored over ten minutes (n = 6). Representative example of three independent experiments.


B Time course of AKT CREB phosphorylation . Melanoma lines were stimulated with 100 nM EDN for 2 - 90 min . AKT CREB phosphorylation expression was evaluated by Western blot . Representative example of three independent experiments .

O O O O B-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O

B Time course of AKT and CREB phosphorylation. Melanoma cell lines were stimulated with 100 nM EDN for 2-90 min. AKT and CREB phosphorylation and expression was evaluated by Western blot. Representative example of three independent experiments.


D High reproducibility in UACC257 A2058 datasets . CV values were grouped according to to represent dataset specific CV distributions . Numbers lines indicate median .

O O O O B-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O O

D High reproducibility in the UACC257 and A2058 datasets. All CV values were grouped according to cell line to represent dataset specific CV distributions. Numbers and lines indicate the median.


A Phosphopeptide MS1 intensities in UACC257 samples were analysed by PCA . Colours indicate experimental groups shading is mapped to time .

O O O B-EXP_ASSAY O B-CELL_LINE O O O O B-EXP_ASSAY O O O O O O O O O O O

A Phosphopeptide MS1 intensities in UACC257 samples were analysed by PCA. Colours indicate experimental groups and shading is mapped to time.


C Comparison of kinetics of 242 shared target phosphopeptides . Log2 FC of EDN / Ctr is plotted from top to bottom for each phosphopeptide for both lines with increases shown in green decreases shown in red .

O O O B-EXP_ASSAY O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O

C Comparison of the kinetics of the 242 shared target phosphopeptides. Log2 FC of EDN/Ctr is plotted from top to bottom for each phosphopeptide for both cell lines with increases shown in green and decreases shown in red.


A , B EDN target phosphopeptides for ( A ) UACC257 ( B ) A2058 were segregated into six clusters each , using c - means clustering ( MFuzz ) . Each represents one phosphopeptide cluster membership is indicated by colour . Most likely kinases for each phosphosite were predicted using NetworKIN enrichment of kinase substrates was calculated for each cluster . Enriched kinases are indicated above each cluster . Note that substrates of these kinases only account for a fraction of phosphosites in a cluster .

O O O B-GENEPROD O O O O O O B-CELL_LINE O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O

A,B EDN target phosphopeptides for (A) UACC257 and (B) A2058 were segregated into six clusters each, using c-means clustering (MFuzz). Each line represents one phosphopeptide and cluster membership is indicated by colour. Most likely kinases for each phosphosite were predicted using NetworKIN and enrichment of kinase substrates was calculated for each cluster. Enriched kinases are indicated above each cluster. Note that substrates of these kinases only account for a fraction of all phosphosites in a cluster.


C Average MS1 z - score kinetics for 217 predicted CaMKII - α sites that are shared between both lines . For comparison , average ( n = 6 ) calcium abundance kinetics are shown .

O O O O O O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O O O

C Average MS1 intensity z-score kinetics for 217 predicted CaMKII-α sites that are shared between both cell lines. For comparison, average (n = 6) calcium abundance kinetics are shown.


Predicted kinases are indicated on top of each panel for which one of their marker substrate phosphorylations was assayed . UACC257 were serum starved stimulated with PBS or EDN at 100 nM for 60 min ( 5 min for CaMKII ) . Ten μg from RIPA lysates were analysed by Western blot . Kinase marker substrate phosphorylations corresponding loading controls are shown . For each kinase , two independent replicates were performed are shown . Each gel also had control lanes with increasing of kinase inhibitors to support phospho band

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Predicted kinases are indicated on top of each panel for which one of their marker substrate phosphorylations was assayed. UACC257 cells were serum starved and stimulated with PBS or EDN at 100 nM for 60 min (5 min for CaMKII). Ten μg protein from RIPA lysates were analysed by Western blot. Kinase marker substrate phosphorylations and corresponding loading controls are shown. For each kinase, two independent replicates were performed and are shown. Each gel also had control lanes with increasing concentration of kinase inhibitors to support phospho band identity


effect of kinase inhibitors on EDN induced migration of UACC257 was analysed using a scratch . Scratches were made in UACC257 monolayers . Serum was removed were treated with PBS or 100 nM EDN in combination with DMSO or kinase inhibitors at 0 . 1 μM or 1 μM in triplicates . A Example pictures showing effect of EDN on migration . free areas are delimited by blue lines . Migration was quantified as change in free between 0 h 48 h . B four inhibitors that were effective at 1 µM are shown in detail , i . e . every point corresponds to one well . C Abundance of E - Cadherin ( normalized to GAPDH Actin ) was assessed by LC - SRM after 48 h treatment with EDN or kinase inhibitors at 1 µM . Only single inhibitor which could reverse EDN induced E - Cadherin repression is shown . * p < 0 . 05 two - sided unpaired t - .

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The effect of kinase inhibitors on EDN induced cell migration of UACC257 cells was analysed using a scratch assay. Scratches were made in UACC257 monolayers. Serum was removed and cells were treated with PBS or 100 nM EDN in combination with DMSO or kinase inhibitors at 0.1 μM or 1 μM in triplicates. A Example pictures showing the effect of EDN on migration. Cell free areas are delimited by blue lines. Migration was quantified as the change in cell free area between 0 h and 48 h. B The four inhibitors that were effective at 1 µM are shown in detail, i.e. every point corresponds to one well. C Abundance of E-Cadherin (normalized to GAPDH and Actin) was assessed by LC-SRM after 48 h treatment with EDN or kinase inhibitors at 1 µM. Only the single inhibitor which could reverse EDN induced E-Cadherin repression is shown. * p < 0.05 two-sided unpaired t-test.


F Clonogenic growth of indicated HPAF - II left untreated or in presence of 150nM LGK974 for 10 days ; representative of three independent replicates .

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F Clonogenic growth assay of indicated HPAF-II cells left untreated or in the presence of 150nM LGK974 for 10 days; representative of three independent replicates.


G Immunoblot of βcatenin expression from lysates of HPAF - II from indicated genotype , treated with vehicle or 100nM LGK974 for 48h ; representative of three biological replicates .

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G Immunoblot of cytoplasmic βcatenin expression from lysates of HPAF-II cells from indicated genotype, treated with vehicle or 100nM LGK974 for 48h; representative of three biological replicates.


A Immunoblot of CCNL1 FBXW7 expression in HPAF - II wild - type FBXW7 - / - , representative blot of 3 independent replicates .

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A Immunoblot of CCNL1 and FBXW7 expression in HPAF-II wild-type and FBXW7-/- cells, representative blot of 3 independent replicates.


B Immunoblot of CCNL1 expression following a cycloheximide chase in HPAF - II wild - type FBXW7 - / - , representative blot of 3 independent replicates . C of cycloheximide chase in B , mean ± of three independent replicates .

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B Immunoblot of CCNL1 expression following a cycloheximide chase in HPAF-II wild-type and FBXW7-/- cells , representative blot of 3 independent replicates. C Quantification of cycloheximide chase in B, mean ± SEM of three independent replicates.


D Immunoblot of CCNL1 expression in HPAF - II wild - type dN - Cul1 expressing , representative blot of 3 independent replicates .

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D Immunoblot of CCNL1 expression in HPAF-II wild-type and dN-Cul1 expressing cells, representative blot of 3 independent replicates.


G Immunoblot of lysates following cycloheximide treatment of HEK293T expressing wild - type or degron - mutated CCNL1 , representative blot of 3 independent replicates . H of cycloheximide chase in G , mean ± of three independent replicates .

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G Immunoblot of lysates following cycloheximide treatment of HEK293T cells expressing wild-type or degron-mutated CCNL1, representative blot of 3 independent replicates. H Quantification of cycloheximide chase in G, mean ± SEM of three independent replicates.


A Immunoblot of immunoprecipitation of FLAG - FBXW7 overexpressed in HEK293T , detecting endogenous CCNL1 . Representative of three independent replicates .

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A Immunoblot of immunoprecipitation of FLAG-FBXW7 overexpressed in HEK293T cells, detecting endogenous CCNL1. Representative of three independent replicates.


B Immunoblot of immunoprecipitation of FLAG - CCNL1 overexpressed in HEK293T , detecting endogenous FBXW7 . Representative of three independent replicates .

B Immunoblot of immunoprecipitation of FLAG-CCNL1 overexpressed in HEK293T cells, detecting endogenous FBXW7. Representative of three independent replicates.


C Immunoblot of ubiquitination demonstrating requirement of 325 - TPALS - 329 degron for ubiquitination of CCNL1 . Representative of three independent replicates .

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C Immunoblot of cellular ubiquitination assay demonstrating the requirement of the 325-TPALS-329 degron for ubiquitination of CCNL1. Representative of three independent replicates.


D Immunoblot of ubiquitination demonstrating requirement of FBXW7 for ubiquitination of CCNL1 . Representative of three independent replicates .

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D Immunoblot of cellular ubiquitination assay demonstrating the requirement of FBXW7 for ubiquitination of CCNL1. Representative of three independent replicates.


E Immunoblot of ubiquitination showing re - expression of FBXW7 in knockout leads to increased CCNL1 ubiquitination . Representative of three independent replicates .

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E Immunoblot of cellular ubiquitination assay showing re-expression of FBXW7 in knockout cells leads to increased CCNL1 ubiquitination. Representative of three independent replicates.


B Immunoblot of HPAF - II wild - type FBXW7 - / - released from overnight nocodazole treatment at indicated time points ,

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B Immunoblot of HPAF-II wild-type and FBXW7-/- released from overnight nocodazole treatment at indicated time points,


D Representative of GFP - tubulin labeled wild - type , FBXW7 - / - , CCNL1OE HPAF - II monitoring progression through cytokinesis following arrest in prometaphase using monastrol ( 150μM for 18 hours ) . E of cytokinetic timing , n = 20 , 25 , 26 ( wild - type , FBXW7 - / , CCNL1OE ) pooled from three independent experiments F Immunoblot of wild - type , FBXW7 - / - CCNL1OE HPAF - II lines demonstrating varying of CCNL1 .

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D Representative images of GFP-tubulin labeled wild-type, FBXW7-/-, and CCNL1OE HPAF-II cells monitoring cellular progression through cytokinesis following arrest in prometaphase using monastrol (150μM for 18 hours). E Quantification of cytokinetic timing, n=20, 25, 26 (wild-type, FBXW7-/, CCNL1OE ) pooled from three independent experiments F Immunoblot of wild-type, FBXW7-/- and CCNL1OE HPAF-II cell lines demonstrating varying levels of CCNL1.


A Clonogenic growth assays for HPAF - II wild - type FBXW7 - / - polyclonal , FBXW7 - / - clones in presence of various OTS964 doses for 14 days . Representative of 4 independent replicates , quantified by crystal violet absorbance at A595 B Clonogenic growth assays for HPAF - II wild - type CCNL1OE polyclonal , CCNL1OE clones in presence of various doses of OTS964 for 14 days . Representative of 4 independent replicates , quantified by crystal violet absorbance at

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A Clonogenic growth assays for HPAF-II wild-type and FBXW7-/- polyclonal, and FBXW7-/- clones in presence of various OTS964 doses for 14 days. Representative images of 4 independent replicates, quantified by crystal violet absorbance at A595 and B Clonogenic growth assays for HPAF-II wild-type and CCNL1OE polyclonal, and CCNL1OE clones in presence of various doses of OTS964 for 14 days. Representative images of 4 independent replicates, quantified by crystal violet absorbance at A595


C Immunoblot for indicated from lysates extracted from FBXW7 - / - CCNL1OE clones .

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C Immunoblot for the indicated proteins from lysates extracted from the individual FBXW7-/- and CCNL1OE clones.


B Immunoblot of lysates from cervical lines demonstrating C33A ( FBXW7R465H ) express high of CCNL1 , representative of three independent replicates .

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B Immunoblot of lysates from cervical cell lines demonstrating C33A cells (FBXW7R465H) express high levels of CCNL1, representative of three independent replicates.


C Clonogenic growth of C33A , Caski , SiHa in presence of various doses of OTS964 for 14 days . Representative of 3 independent replicates , quantified by crystal violet absorbance at

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C Clonogenic growth assay of C33A, Caski, and SiHa cells in presence of various doses of OTS964 for 14 days. Representative images of 3 independent replicates, quantified by crystal violet absorbance at A595


( A ) Detection of VgpA in supernatants from indicated strains by immunoblotting ( IB ) with anti - VgpA antisera .

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(A) Detection of VgpA in supernatants from indicated strains by immunoblotting (IB) with anti-VgpA antisera.


( B ) T3SS2 translocation of VgpA into Caco - 2 using CyaA - ; VopV is a known T3SS2 effector . Error bars represent mean ± standard deviation ( n = 3 biologically independent experiments ) .

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(B) T3SS2 translocation of VgpA into Caco-2 cells using CyaA-based assay; VopV is a known T3SS2 effector. Error bars represent mean ± standard deviation (n=3 biologically independent experiments).


( C ) Localization of translocated VgpA ( upper panel ) VgpAL10A ( lower panel ) during infection .

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(C) Localization of translocated VgpA (upper panel) and VgpAL10A (lower panel) during infection.


( A ) Silver - stained PAGE gel of from a extract that interact with either GST - VgpA or GST glutathione agarose . bound by GST - VgpA ( red arrow ) , but not GST , was identified as EBP2 by mass spectrometry ; GST - VgpA is labeled with a blue arrow .

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(A) Silver-stained PAGE gel of proteins from a nuclear extract that interact with either GST-VgpA or GST glutathione agarose. The protein bound by GST-VgpA (red arrow), but not GST, was identified as EBP2 by mass spectrometry; GST-VgpA is labeled with a blue arrow.


( B ) Western blot using anti - EBP2 as antibody confirmed that EBP2 binds VgpA .

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(B) Western blot using anti-EBP2 as antibody confirmed that EBP2 binds VgpA.


( C ) Interaction of VgpA or VgpA mutants ( VgpAΔ10 VgpAL10A ) with EBP2 .

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(C) Interaction of VgpA or VgpA mutants (VgpAΔ10 and VgpAL10A) with EBP2.


( D ) Localization of VgpA ( upper panel ) or in VgpAL10A ( lower panel ) in HeLa after transfection . EBP2 was detected with anti - EBP2 antibody ( red ) , VgpA VgpAL10A were detected with anti - VgpA sera ( green ) nuclei were stained with DAPI ( blue ) . arrows indicate co - localization of EBP2 VgpA ( upper panel ) . VgpAL10A localizes primarily to nucleoplasm ( arrow , lower panel ) .

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(D) Localization of VgpA (upper panel) or in VgpAL10A (lower panel) in HeLa cells after transfection. EBP2 was detected with anti-EBP2 antibody (red), VgpA and VgpAL10A were detected with anti-VgpA sera (green) and nuclei were stained with DAPI (blue). White arrows indicate co-localization of EBP2 and VgpA (upper panel). VgpAL10A localizes primarily to the nucleoplasm (white arrow, lower panel).


( D ) Localization of VgpA ( upper panel ) or in VgpAL10A ( lower panel ) in HeLa after transfection . EBP2 was detected with anti - EBP2 antibody ( red ) , VgpA VgpAL10A were detected with anti - VgpA sera ( green ) nuclei were stained with DAPI ( blue ) . arrows indicate co - localization of EBP2 VgpA ( upper panel ) . VgpAL10A

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(D) Localization of VgpA (upper panel) or in VgpAL10A (lower panel) in HeLa cells after transfection. EBP2 was detected with anti-EBP2 antibody (red), VgpA and VgpAL10A were detected with anti-VgpA sera (green) and nuclei were stained with DAPI (blue). White arrows indicate co-localization of EBP2 and VgpA (upper panel). VgpAL10A localizes


( A ) of uninfected Caco - 2 or Caco - 2 infected with indicated V . parahaemolyticus strains . were stained with DAPI ( blue ) anti - BrdU antibody ( green ) .

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(A) Fluorescence micrographs of uninfected Caco-2 cells or Caco-2 cells infected with the indicated V. parahaemolyticus strains. Cells were stained with DAPI (blue) and anti-BrdU antibody (green).


( B ) of Caco - 2 transfected with siRNA targeting EBP2 ( siEBP2 ) or control siRNA ( siNC ) subsequently infected with vopZ ' ΔvscN1 . were stained with anti - BrdU antibody ( green ) DAPI ( blue ) .

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(B) Fluorescence micrographs of Caco-2 cells transfected with siRNA targeting EBP2 (siEBP2) or control siRNA (siNC) and subsequently infected with vopZ'ΔvscN1. Cells were stained with anti-BrdU antibody (green) and DAPI (blue).


( C ) of BrdU - positive ( green ) among total ( green + blue ) was analyzed for 100 in three experiments . Error bars represent mean ± standard deviation ( n = 3 biologically independent experiments ) . One - way of variance ( ANOVA ) was used for statistical . * P < 0 . 05 ( when compared to uninfected Caco - 2 ) .

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(C) The percentage of BrdU-positive cells (green) among the total cells (green + blue) was analyzed for 100 cells in three experiments. Error bars represent mean ± standard deviation (n=3 biologically independent experiments). One-way analysis of variance (ANOVA) was used for statistical analysis. *P<0.05 (when compared to the uninfected Caco-2 cells).


( D ) of BrdU + was analyzed as above for treated with siNC or siEBP2 . Error bars represent mean ± standard deviation ( n = 3 biologically independent experiments ) . Two - way ANOVA was used for statistical . * P < 0 . 05 ( when compared to siNC - transfected that are infected with vopZ ' ΔvscN1 ) .

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(D) The percentage of BrdU+ cells was analyzed as above for cells treated with siNC or siEBP2. Error bars represent mean ± standard deviation (n=3 biologically independent experiments). Two-way ANOVA was used for statistical analysis. *P<0.05 (when compared to siNC-transfected cells that are infected with vopZ'ΔvscN1).


( E ) Immunoblot of EBP2 Actin in Caco - 2 transfected with siEBP2 or siNC .

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(E) Immunoblot of EBP2 and Actin in Caco-2 cells transfected with siEBP2 or siNC.


( A ) Localization of c - Myc in uninfected Caco - 2 ( first row ) , Caco - 2 infected with V . parahaemolyticus vopZ ' ΔvscN1 ( wild type VgpA , second row ) , Caco - 2 infected with vopZ ' ΔvscN1vgpA ' ( third row ) , Caco - 2 infected with vopZ ' ΔvscN1vgpA ' : pvgpA ( fourth row ) Caco - 2 infected with vopZ ' ΔvscN1vgpA ' : pvgpAL10A ( fifth row ) . Fixed were stained with DAPI ( blue ) , anti - EBP2 ( red ) , anti c - Myc ( green ) antibodies . arrows are examples indicating co - localization of EBP2 c - Myc .

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(A) Localization of c-Myc in uninfected Caco-2 cells (first row), Caco-2 cells infected with V. parahaemolyticus vopZ'ΔvscN1 (wild type VgpA, second row), Caco-2 cells infected with vopZ'ΔvscN1vgpA' (third row), Caco-2 cells infected with vopZ'ΔvscN1vgpA':pvgpA (fourth row) and Caco-2 cells infected with vopZ'ΔvscN1vgpA':pvgpAL10A (fifth row). Fixed cells were stained with DAPI (blue), and anti-EBP2 (red), and anti c-Myc (green) antibodies. White arrows are examples indicating the co-localization of EBP2 and c-Myc.


( B Detection of c - Myc or EBP2 in extracts from uninfected Caco - 2 or after infection with indicated strains . Following infection , extracts were obtained loaded on A / G beads cross - linked with anti - EBP2 ( B , IP : anti - EBP2 ) Input eluates were blotted with anti - EBP2 , anti - c - Myc or anti - VgpA antibodies , respectively . Quantified value ( mean ± standard deviation ) as measured by band ( normalized to uninfected ) from three independent experiments are presented under signals of blot of IP samples . n . d . , not determined .

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(B Detection of c-Myc or EBP2 in nuclear extracts from uninfected Caco-2 cells or after infection with indicated strains. Following infection, nuclear extracts were obtained and loaded on protein A/G beads cross-linked with anti-EBP2 (B, IP: anti-EBP2) Input and eluates were blotted with anti-EBP2, anti-c-Myc or anti-VgpA antibodies, respectively. Quantified value (mean ± standard deviation) as measured by band intensity (normalized to uninfected) from three independent experiments are presented under the signals of the blot of IP samples. n.d., not determined.


C ) Detection of c - Myc or EBP2 in extracts from uninfected Caco - 2 or after infection with indicated strains . Following infection , extracts were obtained loaded on A / G beads cross - linked anti - C - Myc ( C , IP : anti - c - Myc ) antibodies , respectively . Input eluates were blotted with anti - EBP2 , anti - c - Myc or anti - VgpA antibodies , respectively . Quantified value ( mean ± standard deviation ) as measured by band ( normalized to uninfected ) from three independent experiments are presented under signals of blot of IP samples . n . d . , not determined .

O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O

C) Detection of c-Myc or EBP2 in nuclear extracts from uninfected Caco-2 cells or after infection with indicated strains. Following infection, nuclear extracts were obtained and loaded on protein A/G beads cross-linked anti-C-Myc (C, IP: anti-c-Myc) antibodies, respectively. Input and eluates were blotted with anti-EBP2, anti-c-Myc or anti-VgpA antibodies, respectively. Quantified value (mean ± standard deviation) as measured by band intensity (normalized to uninfected) from three independent experiments are presented under the signals of the blot of IP samples. n.d., not determined.


( D ) Purified EBP2 , c - Myc GST - VgpA or GST - VgpAL10A were mixed then pulled down with A / G beads cross - linked with anti - EBP2 . Input eluates were blotted with anti - EBP2 , anti - c - Myc or anti - VgpA antibodies , respectively . Quantified value ( mean ± standard deviation ) as measured by band ( normalized to EBP2 + c - Myc sample without GST - VgpA GST - VgpAL10A ) from three independent experiments are presented under signals of blot of IP samples . n . d . , not determined .

O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O

(D) Purified proteins EBP2, c-Myc and GST-VgpA or GST-VgpAL10A were mixed and then pulled down with protein A/G beads cross-linked with anti-EBP2. Input and eluates were blotted with anti-EBP2, anti-c-Myc or anti-VgpA antibodies, respectively. Quantified value (mean ± standard deviation) as measured by band intensity (normalized to EBP2+c-Myc sample without GST-VgpA and GST-VgpAL10A) from three independent experiments are presented under the signals of the blot of IP samples. n.d., not determined.


( E ) Caco - 2 were infected with indicated strains treated with cycloheximide ( CHX ) for indicated time . Whole lysates were analyzed by western blot using anti - c - Myc anti - Actin antibody .

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O

(E) Caco-2 cells were infected with the indicated strains and treated with cycloheximide (CHX) for the indicated time. Whole cells lysates were analyzed by western blot using anti-c-Myc and anti-Actin antibody.


( A ) Amounts of rRNA were from uninfected Caco - 2 or infected with indicated strains . * P < 0 . 05 ( when compared to uninfected Caco - 2 ) .

O O O O O B-SMALL_MOLECULE O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O

(A) Amounts of rRNA were from uninfected Caco-2 cells or cells infected with indicated strains. *P<0.05 (when compared to the uninfected Caco-2 cells).


( B ) Amounts of rRNA in uninfected or infected Caco - 2 transfected with control ( siNC ) or EBP2 - targeting ( siEBP2 ) siRNAs . * P < 0 . 05 ( when compared to siNC - transfected that are infected with vopZ ' ΔvscN1 ) .

O O O O O B-SMALL_MOLECULE O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O

(B) Amounts of rRNA in uninfected or infected Caco-2 cells transfected with control (siNC) or EBP2-targeting (siEBP2) siRNAs. *P<0.05 (when compared to the siNC-transfected cells that are infected with vopZ'ΔvscN1).


( C ) Amounts of polymerase I ( POLR1A ) were from uninfected Caco - 2 or infected with indicated strains . * P < 0 . 05 ( when compared to uninfected Caco - 2 ) .

O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O

(C) Amounts of RNA polymerase I (POLR1A) were from uninfected Caco-2 cells or cells infected with indicated strains. *P<0.05 (when compared to the uninfected Caco-2 cells).


( D ) . Amounts of rRNA in intestinal homogenates from infant rabbits infected with indicated strains 18 38 h after infection . * P < 0 . 05 ( when compared to uninfected rabbits at 18 hours ) . ΔP < 0 . 05 ( when compared to uninfected rabbits at 38 hours post infection ) .

O O O O O O B-SMALL_MOLECULE O B-TISSUE O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O

(D). Amounts of rRNA in intestinal homogenates from infant rabbits infected with indicated strains 18 and 38 h after infection. *P<0.05 (when compared to the uninfected rabbits at 18 hours). ΔP<0.05 (when compared to the uninfected rabbits at 38 hours post infection).


( A ) VgpA ( upper panel : infected with WT ) VgpAL10A ( lower panel : infected with vgpAL10A ) localization in intestinal epithelial of infected . Arrows in upper panel indicate VgpA localized as in nucleus ; arrow in lower panel indicates VgpAL10A localized diffusively in nucleoplasm .

O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O B-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE O O O O O O O O B-GENEPROD B-EXP_ASSAY O O B-SUBCELLULAR O O O O O O B-GENEPROD B-EXP_ASSAY O O B-SUBCELLULAR O

(A) VgpA (upper panel: animals infected with WT) and VgpAL10A (lower panel: animals infected with vgpAL10A) localization in intestinal epithelial cells of infected animals. Arrows in upper panel indicate VgpA localized as punctae in the nucleus; the arrow in the lower panel indicates VgpAL10A localized diffusively in the nucleoplasm.


( B ) VgpA VgpAL10A interactions with EBP2 in intestinal epithelial isolated from infant rabbits infected with WT or vgpAL10A V . parahaemolyticus . Inputs represent detected in intestinal epithelial of uninfected rabbits or rabbits infected with indicated strains . Outputs represent eluates from A / G beads cross - linked with anti - EBP2 ( IP : anti - EBP2 ) or anti - VgpA ( IP : anti - VgpA ) antibodies analyzed with anti - VgpA anti - EBP2 antibodies .

O O O B-GENEPROD I-GENEPROD B-EXP_ASSAY O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O B-ORGANISM O O O O B-GENEPROD B-ORGANISM I-ORGANISM I-ORGANISM O O O O O B-CELL_TYPE I-CELL_TYPE O O B-ORGANISM O B-ORGANISM O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O B-GENEPROD O O B-GENEPROD O O

(B) VgpA and VgpAL10A interactions with EBP2 in intestinal epithelial cells isolated from infant rabbits infected with WT or vgpAL10A V. parahaemolyticus. Inputs represent proteins detected in intestinal epithelial cells of uninfected rabbits or rabbits infected with the indicated strains. Outputs represent eluates from protein A/G beads cross-linked with anti-EBP2 (IP: anti-EBP2) or anti-VgpA (IP: anti-VgpA) antibodies and analyzed with anti-VgpA and anti-EBP2 antibodies.


( C ) Frozen sections from uninfected rabbits or rabbits infected with indicated strains for 18 hours were stained with anti - Ki67 antibodies to detect actively dividing ( green ) , phalloidin to visualize F - actin ( red ) DAPI to detect nuclei ( blue ) .

O O O O O O O B-ORGANISM O B-ORGANISM O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O B-SMALL_MOLECULE O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O B-SMALL_MOLECULE O O B-SUBCELLULAR O O O O

(C) Frozen sections from uninfected rabbits or rabbits infected with the indicated strains for 18 hours were stained with anti-Ki67 antibodies to detect actively dividing cells (green), phalloidin to visualize F-actin (red) and DAPI to detect nuclei (blue).


( D ) Eighteen hours after infant rabbits were infected with WT ( middle panel ) or vgpAL10A ( lower panel ) V . parahaemolyticus , fixed sections from small intestine were stained with antibodies to c - Myc ( green ) , EBP2 ( red ) , DAPI ( blue ) . Uninfected rabbits were processed similarly were included as a control ( upper panel ) . arrows indicate co - localization of c - Myc EBP2 ( middle panel ) .

O O O O O O O B-ORGANISM O O O O O O O O O B-GENEPROD O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O B-TISSUE I-TISSUE O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O B-ORGANISM O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O

(D) Eighteen hours after infant rabbits were infected with WT (middle panel) or vgpAL10A (lower panel) V. parahaemolyticus, fixed sections from the small intestine were stained with antibodies to c-Myc (green), EBP2 (red), and DAPI (blue). Uninfected rabbits were processed similarly and were included as a control (upper panel). White arrows indicate co-localization of c-Myc and EBP2 (middle panel).


( A ) CFU in homogenates from distal small intestines of infant rabbits infected with indicated strains 18 , 28 38 h following inoculation . Black horizontal indicates mean CFU ( n = 7 - 10 / group ) . One - way ANOVA was used for statistical . * P < 0 . 05 for indicated comparison .

O O O B-EXP_ASSAY O O O O B-TISSUE I-TISSUE O O B-ORGANISM O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) CFU in homogenates from the distal small intestines of infant rabbits infected with the indicated strains 18, 28 and 38 h following inoculation. Black horizontal line indicates mean CFU (n=7-10 animals/group). One-way ANOVA was used for statistical analysis. *P<0.05 for the indicated comparison.


( B ) H & E stained sections from distal small intestine from rabbits infected with indicated strains 18 hours after infection . Black arrows point to colonies closely attached to epithelium .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-TISSUE I-TISSUE O B-ORGANISM O O O O O O O O O O O O O O O O O B-TISSUE O

(B) H&E stained sections from the distal small intestine from the rabbits infected with the indicated strains 18 hours after infection. Black arrows point to bacterial colonies closely attached to the epithelium.


( C ) Fluid accumulation in rabbits infected with indicated strains 38 hours post inoculation . Error bars represent mean ± standard deviation ( n = 7 / group ) . One - way ANOVA was used for statistical . * P < 0 . 05 ( when compared to rabbits infected with WT ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O

(C) Fluid accumulation in rabbits infected with the indicated strains 38 hours post inoculation. Error bars represent mean ± standard deviation (n=7 animals/group). One-way ANOVA was used for statistical analysis. *P<0.05 (when compared to the rabbits infected with WT).


C , AltFUS ( arrow ) endogenous expression in Human tissues ( brain , kidney - 100 μg ) , in HEK293 HeLa cultured ( 100 μg ) using over - expression of altFUS CDS in HEK293 ( 50 μg ) as positive control ( representative image from n = 3 ) . asterisk indicates a species detected with anti - altFUS antibody specifically in brain .

O O B-GENEPROD O O O O B-EXP_ASSAY O B-ORGANISM O O B-TISSUE O B-TISSUE O O O O O O B-CELL_LINE I-CELL_LINE O O O O O O O O O O B-GENEPROD O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-TISSUE O

C, AltFUS (arrow) endogenous expression in Human tissues (brain, muscles and kidney - 100 μg), in HEK293 and HeLa cultured cells (100 μg) and using the over-expression of altFUS CDS in HEK293 cells (50 μg) as positive control (representative image from n=3). The asterisk indicates a protein species detected with the anti-altFUS antibody specifically in the brain.


AltFUS ( arrow ) endogenous expression in motor cortex of three C9orf72 three sporadic ALS ( D ) ( representative image from n = 3 ) . asterisk indicates a species detected with anti - altFUS antibody specifically in brain .

B-GENEPROD O O O O B-EXP_ASSAY O B-TISSUE I-TISSUE O O O O B-DISEASE I-DISEASE O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-TISSUE O

AltFUS (arrow) endogenous expression in the motor cortex of three C9orf72 and three sporadic ALS patients (D) (representative image from n=3). The asterisk indicates a protein species detected with the anti-altFUS antibody specifically in the brain.


AltFUS ( arrow ) endogenous expression in iPSC - derived motor neurons of three lines from controls from ALS ( E ) ( representative image from n = 3 ) . asterisk indicates a species detected with anti - altFUS antibody specifically in brain .

B-GENEPROD O O O O B-EXP_ASSAY O B-CELL_TYPE O O B-CELL_TYPE I-CELL_TYPE O O O O O O B-DISEASE O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-TISSUE O

AltFUS (arrow) endogenous expression in iPSC-derived motor neurons of three lines from controls and from ALS patients (E) (representative image from n=3). The asterisk indicates a protein species detected with the anti-altFUS antibody specifically in the brain.


I , Endogenous altFUS mitochondrial expression in HEK293 following fractionation ( representative image from n = 3 ) , with Tubulin as a marker of fraction VDAC as a marker of mitochondrial fraction . We used siFUS transfected as a negative control altFUS transfected as a positive control for altFUS expression ( WCL = Whole Lysate , Cyto = Cytosol fraction , Mito = mitochondrial fraction ) .

O O O B-GENEPROD B-SUBCELLULAR B-EXP_ASSAY O B-CELL_LINE O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O B-SUBCELLULAR O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD B-EXP_ASSAY O O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR O O B-SUBCELLULAR O B-SUBCELLULAR O O O

I, Endogenous altFUS mitochondrial expression in HEK293 cells following fractionation (representative image from n=3), with Tubulin as a marker of the cytosolic fraction and VDAC as a marker of the mitochondrial fraction. We used siFUS transfected cells as a negative control and altFUS transfected cells as a positive control for altFUS expression (WCL = Whole Cell Lysate, Cyto = Cytosol fraction, Mito = mitochondrial fraction).


J , Representative of mitochondrial network ( Tomm20 in red ) in mock altFUS - Flag ( green ) transfected HeLa ( n = 3 ) . scale bar corresponds to 10μm . K , Proportion of tubules globules in mitochondrial network of mock HeLa HeLa transfected with altFUS - Flag was done over a minimum of 100 across a technical duplicate per independent experiments ( n = 3 , i . e . a minimum of 300 per biological conditions , p - value < 0 . 001 , Mann - Whitney U ) . boxes extend to 25th 75th percentiles , with median marked . whiskers correspond to 5th 95th percentiles .

O O O O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O B-CELL_LINE O O O O O O O O O O O O O O O O B-TISSUE I-TISSUE O B-SUBCELLULAR I-SUBCELLULAR O O B-CELL_LINE I-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

J, Representative images of the mitochondrial network (Tomm20 in red) in mock and altFUS-Flag (green) transfected HeLa cells (n=3). The white scale bar corresponds to 10μm. K, Proportion of tubules and globules in the mitochondrial network of mock HeLa cells and HeLa cells transfected with altFUS-Flag Quantification was done over a minimum of 100 cells across a technical duplicate per independent experiments (n=3, i.e. a minimum of 300 cells per biological conditions, p-value < 0.001, Mann-Whitney U test). The boxes extend to the 25th and 75th percentiles, with the median marked. The whiskers correspond to the 5th and 95th percentiles.


E , Scatter plot of identified by AP - MS indicating their enrichment ( Fold - change over control ) their SAINT probability score . above 0 . 8 threshold ( grey ) are indicated in black , others in grey . AltFUS is indicated in red ( bait ) preys known to regulate autophagy or stress response are indicated in green .

O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O

E, Scatter plot of the proteins identified by AP-MS indicating their enrichment (Fold-change over control) and their SAINT probability score. Proteins above the 0.8 threshold (grey line) are indicated in black, others in grey. AltFUS is indicated in red (bait) and preys known to regulate the autophagy or the cellular stress response are indicated in green.


B , LC3 - II accumulation after bafilomycin treatment from mock , altFUS , FUS , FUS ( Ø ) , FUS - R495x , FUS ( Ø ) - R495x transfected FUS ( Ø ) - R495x altFUS co - transfected across 3 independent experiments ( n = 3 , mean ± SD ) . corresponds to treated / untreated of LC3 - II abundance Statistical significance is relative to mock condition unless otherwise indicated ( * * * * = p value < 0 . 0001 , * * * = p value < 0 . 001 , * * = p value < 0 . 01 , n . s . = non - significant , two - way ANOVA with Tukey ' s multiple comparison correction ) .

O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-SMALL_MOLECULE O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B, LC3-II accumulation after bafilomycin treatment from mock, altFUS, FUS, FUS(Ø), FUS-R495x, FUS(Ø)-R495x transfected cells and FUS(Ø)-R495x and altFUS co-transfected cells across 3 independent experiments (n=3, mean ± SD). The quantification corresponds to the treated/untreated ratio of LC3-II abundance Statistical significance is relative to the mock condition unless otherwise indicated (**** = p value < 0.0001, *** = p value < 0.001, ** = p value < 0.01, n.s. = non-significant, two-way ANOVA with Tukey's multiple comparison correction).


D - E , of FUS granules , number ( D ) ( μm2 ) ( E ) in over - expressing bicistronic ( + ) or monocistronic ( - ) construct for FUS , FUS - G156E , FUS - R495x , FUS - K510E , FUS - Q519x , FUS - Q519I - fs527x , FUS - R521C , FUS - P525L . Statistical comparisons are made between bicistronic monocistronic versions of each construct ( n = 3 - biological replicates , mean ± SD , * * * * = p value < 0 . 0001 , * * * = p value < 0 . 001 , * * = p value < 0 . 01 , * = p value < 0 . 05 , n . s . = non - significant , one - way ANOVA with Sidak ' s multiple comparison ) .

O O O O O B-GENEPROD B-SUBCELLULAR O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D-E, Quantification of FUS cytoplasmic granules, number (D) and area (μm2) (E) in cells over-expressing the bicistronic (+) or monocistronic (-) construct for FUS, FUS-G156E, FUS-R495x, FUS-K510E, FUS-Q519x, FUS-Q519I-fs527x, FUS-R521C, and FUS-P525L. Statistical comparisons are made between bicistronic and monocistronic versions of each construct (n=3 - biological replicates, mean ± SD, **** = p value < 0.0001, *** = p value < 0.001, ** = p value < 0.01, * = p value < 0.05, n.s. = non-significant, one-way ANOVA test with Sidak's multiple comparison).


C - E , Locomotion represented by of climbing success in control RU - 486 - treated transgenic Drosophila expressing mCherry or altFUS ( C ) , bicistronic FUS or monocistronic FUS ( Ø ) ( D ) , bicistronic FUS - R495x or monocistronic FUS ( Ø ) - R495x ( E ) at day 1 , 10 20 post - induction . Statistical comparisons were made between each population ( n = 4 - biological replicates ) . Indicated significance is between monocistronic bicistronic transgenic flies of RU - 486 - treated population ( mean ± SD , n . s . = non - significant , * = p value < 0 . 05 , * * * = p value < 0 . 001 , two - way ANOVA with Tukey ' s multiple comparison correction ) .

O O O O B-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-ORGANISM O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C-E, Locomotion assay represented by the percentage of climbing success in control and RU-486-treated transgenic Drosophila expressing mCherry or altFUS (C), the bicistronic FUS or the monocistronic FUS(Ø) (D), and the bicistronic FUS-R495x or the monocistronic FUS(Ø)-R495x (E) at day 1, 10 and 20 post-induction. Statistical comparisons were made between each population (n=4 - biological replicates). Indicated significance is between the monocistronic and the bicistronic transgenic flies of the RU-486-treated population (mean ± SD, n.s. = non-significant, * = p value < 0.05, *** = p value < 0.001, two-way ANOVA with Tukey's multiple comparison correction).


A , Graphical representation of FUS synonymous mutations ( yellow ) found in ALS . canonical FUS mRNA is represented in blue ( ENST00000254108 or NM _ 004960 ) . FUS coding is indicated in blue , altFUS coding is indicated in green . B , by confocal microscopy of TDP - 43 ( ) , FUS ( GFP tagged - green ) altFUS ( Flag tagged - red ) in HeLa over - expressing GFP - FUS ( Flag ) , GFP - FUS ( P31L - Flag ) - S44 = , GFP - FUS ( A38V - Flag ) - G51 = , GFP - FUS ( A46V - Flag ) - G59 = , GFP - FUS ( R64P - Flag ) - S77 = ( representative from n = 3 ) . arrows indicate some TDP - 43 . scale bar corresponds to 10 μm . C , of with TDP - 43 in HeLa ( see panel B ) . data are represented as fold - change compared to GFP - FUS ( Flag ) expressing . Statistical significance is indicated above bars ( n = 3 - biological replicates , mean ± SD , * * * * = p value < 0 . 0001 , two - way ANOVA with Tukey ' s multiple comparison correction ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_LINE O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A, Graphical representation of FUS synonymous mutations (yellow) found in ALS patients. The canonical FUS mRNA is represented in dark blue (ENST00000254108 or NM_004960). The FUS protein coding sequence is indicated in light blue, and the altFUS protein coding sequence is indicated in green. B, Images by confocal microscopy of TDP-43 (white), FUS (GFP tagged - green) and altFUS (Flag tagged - red) in HeLa cells over-expressing GFP-FUS(Flag), GFP-FUS(P31L-Flag)-S44=, GFP-FUS(A38V-Flag)-G51=, GFP-FUS(A46V-Flag)-G59=, GFP-FUS(R64P-Flag)-S77= (representative images from n=3). White arrows indicate some TDP-43 aggregates. The white scale bar corresponds to 10 μm. C, Quantification of cells with TDP-43 aggregates in HeLa cells (see panel B). The data are represented as the fold-change compared to the GFP-FUS(Flag) expressing cells. Statistical significance is indicated above the bars (n=3 - biological replicates, mean ± SD, **** = p value < 0.0001, two-way ANOVA with Tukey's multiple comparison correction).


A . Representative pseudo colour of sypHy in WT BsnGT hippocampal neurons at , upon stimulation with 40 900 APs at 20Hz ( in presence of bafilomycin A1 ) upon application of NH4Cl to visualise SVs that were refractory to electrical stimulation . B . Average traces of normalised change ( ΔF / F0 ) of sypHy in WT ( black ) BsnGT ( red ) neurons as described in A . Intensities were normalized to peak of NH4Cl response . C . Quantifications of mean RRP fraction in WT BsnGT . D . Frequency distribution histograms of response amplitudes of 1050 synaptic ( from 6 independent experiments ) to stimulation with 40 AP . distribution is shifted to lower values in BsnGT . E . Quantifications of mean TRP fraction in WT BsnGT . F . Frequency distribution histograms of synaptic response amplitudes in BsnGT WT neurons stimulated with 900 AP . Note shift in distribution between genotypes . Data information : In plots , interquartile range median are depicted as boxes , minimal maximal values as whiskers , + indicates mean . In frequency distribution histograms ( D F ) black lines depict superimposed Gaussian fits for each group . sample n ( in parentheses ) corresponds to number of analysed imaging experiments ( B , C , E ) In D F , data of 1050 synapses per genotype were processed . Data is obtained from 6 ( A - F ) independent culture preparations . Significance was assessed with Student ' s t - ; * * * p < 0 . 001 . Scale bar is 2 µm in

O O O O O O B-GENEPROD O O B-GENEPROD B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O B-SUBCELLULAR O O O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Representative pseudo colour images of sypHy fluorescence in WT and BsnGT hippocampal neurons at rest, upon stimulation with 40 and 900 APs at 20Hz (in the presence of bafilomycin A1) and upon application of NH4Cl to visualise SVs that were refractory to electrical stimulation. B. Average traces of the normalised fluorescence change (ΔF/F0) of sypHy in WT (black) and BsnGT (red) neurons as described in A. Intensities were normalized to the peak of NH4Cl response. C. Quantifications of mean RRP fraction in WT and BsnGT. D. Frequency distribution histograms of the response amplitudes of 1050 individual synaptic puncta (from 6 independent experiments) to stimulation with 40 AP. The distribution is shifted to lower values in BsnGT. E. Quantifications of mean TRP fraction in WT and BsnGT. F. Frequency distribution histograms of synaptic response amplitudes in BsnGT and WT neurons stimulated with 900 AP. Note the shift in distribution between genotypes. Data information: In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers, and + indicates mean. In the frequency distribution histograms (D and F) black lines depict superimposed Gaussian fits for each group. The sample size n (in parentheses) corresponds to the number of analysed imaging experiments (B, C, E) In D and F, the data of 1050 synapses per genotype were processed. Data is obtained from 6 (A-F) independent culture preparations. Significance was assessed with Student's t-test; *** p < 0.001. Scale bar is 2 µm in A