EMBO/soda-nel huggingface dataset:
P-tau235: a novel biomarker for staging preclinical Alzheimer's disease (2021)
Figure 5

Figure 5. CSF biomarkers trajectories in preclinical AD. Trajectories of the different tau biomarkers in preclinical AD using a local weighted regression method (Loess curve). Changes on biomarkers levels in CSF are represented as z-scores using Aβ PET in centiloid scale (CL) as a proxy of disease progression. Abnormal biomarker levels were determined as two standard deviations (SD) above the mean. Incipient Aβ pathology positivity was determined as Aβ PET higher than CL 12 (Mila-Aloma et al., 2020). All samples were run in singlicates.
P-tau235: a novel biomarker for staging preclinical Alzheimer's disease (2021)
Figure 6

Figure 6. CSF p-tau231 and p-tau235 sequential phosphorylation in ALFA+ (preclinical AD). (A) Stacked bar chart depicting the percentages of [231+/235+], [231+/235-], [231-/235-] and [231-/235+] cases in each preclinical group. Table below indicates the exact percentages of participants in each group (exact number of participants in parenthesis). Discordant cases with the sequential phosphorylation hypothesis [231-/+235] shown in grey. (B) Correlation between CSF p-tau235 and p-tau231 in the whole ALFA+ cohort (Spearman's rank correlation: rS = 0.80, P˂0.0001). Assay cut-offs were determined as the mean + 2 SD of the A-T- group (defining p-tau231 and p-tau235 positivity or negativity in each participant). Cut-off values are indicated in red (19.92 and 9.59 pg/mL for CSF p-tau235 and p-tau231 respectively) and displayed with black dashed lines, resulting in four quadrants, each of them representing the four different positive or negatively status for each biomarker. Discordant cases with the sequential phosphorylation hypothesis ([231-/+235]) showed on the lower right quadrant (2.35% of the total, 9 participants out 383). All samples were run in singlicates.
Pseudomonas aeruginosa survives in epithelia by ExoS-mediated inhibition of autophagy and mTOR (2020)
Figure 1

Figure 1. P. aeruginosa T3SS protected P. aeruginosa from autophagy elimination. A549 cells stably expressing lentivirus vector containing random shRNA or Atg7 specific shRNA were generated.

(A) Cells were incubated for 2 hours in the presence or absence of 50 nM of lysosomal inhibitor chloroquine (CQ). Cell lysates were evaluated by immunoblotting.

(B) Cells were infected, for 1 and 4 hours, with K. pneumonia at MOI of 10 followed by 1 hour of gentamycin treatment. Cell lysates were plated in LB agar for 18 hours and CFU were determined.

(C) Schematic of WT P. aeruginosa and ΔpscD P. aeruginosa mutant.

(D-E) Cells were infected, for 1-4 hours, with WT (D) or ΔpscD P. aeruginosa (E) at MOI of 10. All infections were followed by 1 hour of gentamycin treatment. Cell lysates were plated in LB agar for 18 hours and CFU were determined. Data information: In (B) and (D-E), the triangles and dots represent the individual test (three technical replicates per individual test) on control and Atg7 knock down cells. The bars represent the mean CFU

of each group (three biological replicates each group), error bars represent standard

deviation. Statistical significance between assays were calculated using two-tailed student's t test with Welch's correction. NS: not significant; *p ≤ 0.05; **p ≤ 0.01.
Pseudomonas aeruginosa survives in epithelia by ExoS-mediated inhibition of autophagy and mTOR (2020)
Figure 2

Figure 2. P. aeruginosa inhibited autophagy by a T3SS-dependent mechanism.

(A) A549 cells were infected with WT P. aeruginosa for various time points and cell lysates were evaluated by immunoblotting.

(B) A549 cells infected with WT or ΔpscD P. aeruginosa for 4 hours or left uninfected (NI) in the presence of lysosomal inhibitor CQ (50 nM). The cell lysates were evaluated by immunoblotting.

(C) A549 cells stably expressing GFP-LC3 were left non-infected or infected, for 4 hours, with WT or ΔpscD P. aeruginosa, and evaluated by fluorescence microscopy. Cell nuclei were counterstained with DAPI (blue) and autophagosome were visualized as green puncta. Graph represents quantitative analysis of GFP-LC3 positive autophagosome per cell, scale bars: 10 µm. Each bar represents mean value of three independent experiments, error bars represent standard deviation. Statistical significance between assays was determined using One-way ANOVA followed by Dunn's Multiple Comparison Test. **p ≤ 0.01.

(D-E) A549 cells were treated for 16 hours with rapamycin 0.8 μg/ml or DMSO, then infected with either WT (D) or ΔpscD P. aeruginosa and followed by 1 hour of gentamycin treatment (E). The circles and squares represent the individual test (three technical replicates per individual test) with DMSO and rapamycin treatment. The bars represent the means of CFU from three independent experiments, error bars represent standard deviation. The significance of differences between different drug treatment was determined using two-tailed student's t test with Welch's correction. NS: not significant; *p ≤ 0.05; **p ≤ 0.01.
Pseudomonas aeruginosa survives in epithelia by ExoS-mediated inhibition of autophagy and mTOR (2020)
Figure 3

Figure 3. The P. aeruginosa ExoS was required and sufficient for autophagy inhibition. ExoS protected P. aeruginosa from autophagy elimination.

(A,B) (A) A549 cells or (B) primary human bronchial epithelial cells were infected with one of several P. aeruginosa mutants. Cell lysates were evaluated by immunoblotting.

(C) A549 cells, stably expressing GFP-LC3, were infected with WT or P. aeruginosa mutants and evaluated by fluorescence microscopy, scale bars: 10 µm. GFP-LC3 puncta representing the autophagosomes was quantified and presented in the graph. Each bar represents mean value from three independent experiments and error bars represent standard deviation. Statistical significance between assays was determined using One-way ANOVA followed by Dunn's Multiple Comparison Test. *p ≤ 0.05; **p ≤ 0.01. P. aeruginosa mutant strains legend: ΔSTY (mutant deficient for the three cytotoxins), WT (contains all cytotoxin), ΔS (deficient for ExoS but still express ExoT and ExoY), ΔTY (express ExoS but deficient for ExoT and ExoY).

(D,E) A549 cells, stably expressing GFP-LC3, were infected for 4 hours with WT or with one of P. aeruginosa mutants (ΔSTY, ΔS and ΔTY) and evaluated by fluorescence microscopy (D). P. aeruginosa were visualized using specific antibody (red) and the colocalized bacteria signals with GFP-LC3 puncta (green) were merged and presented as yellow. Scale bars: 10 µm. (E) Quantitative analysis of co-localization of internalized P. aeruginosa with GFP-LC3 positive puncta. The colocalization of these two signals was quantified with 10 independent visual fields of more than 100 cells. Data represent mean ± SD of three independent experiments. Statistical significance between assays was determined using One-way ANOVA followed by Dunn's Multiple Comparison Test. *p ≤ 0.05; **p ≤ 0.01.

(F) Representative transmission electron microscopy images of A549 cells infected with P. aeruginosa containing ExoS (ΔTY) or deficient for the three T3SS effector proteins (ΔSTY) for 4 hours. ΔSTY P. aeruginosa (white star) was engulfed by multiple membrane autophagosomes (black arrow). ΔTY P. aeruginosa (white star) were found in cytoplasm with single membrane surrounded, scale bars: 0.5 µm.

(G) A549 cells, stably expressing lentiviral vectors containing random shRNA or Atg7 shRNA, were infected with WT or P. aeruginosa mutants (ΔSTY, ΔTY and ΔS) at MOI of 10. All infections were followed by 1 hours of gentamycin treatment. Cell lysates were plated in LB agar for 18 hours and CFU were determined. Data information: The triangles and dots represent the individual test (three technical replicates per individual test) on control and Atg7 knock down cells from three biological replicates. The bars represent the means of CFU of each group (three biological replicates each group), error bars represent standard deviation. The significance of differences between different assay was determined using two-tailed student's t test with Welch's correction, NS: not significant; **p ≤ 0.01.
Pseudomonas aeruginosa survives in epithelia by ExoS-mediated inhibition of autophagy and mTOR (2020)
Figure 4

Figure 4. ExoS protected P. aeruginosa from autophagy elimination in vivo.

(A) Primary airway epithelial cells were isolated from tracheas of Atg7+/+ and Atg7-/- mouse and were evaluated by immunoblotting.

(B) Sections of tracheas from Atg7+/+ and Atg7-/- mouse were evaluated by immunofluorescence microscopy following staining of nuclear DNA with DAPI (Blue), ciliated epithelial cell marker β-tubulin IV (green) and Atg7 (red). Scale bars: 20 µm.

(C,D) Atg7+/+ and Atg7-/- mice were intranasally injected with 3 x107 CFU of WT, ΔS, ΔTY or ΔSTY P. aeruginosa. After 24 hours, lungs (C), spleens and livers (D) were harvested, homogenized and diluted for CFU counting. Each data point represents bacterial count (Log10 CFU/g) from individual mouse. The horizontal lines represent the mean value from each group (6 mice each group), error bars represent standard deviation. The significance of differences between different drug treatment was determined using One-way ANOVA and Dunn's multiple comparisons test., NS: not significant; *p ≤ 0.05

(E,F) Atg7+/+ and Atg7-/- mice were intranasally injected with PBS with or without 1 x109 CFU of ΔTY (E) ΔSTY (F). Mice survival was monitored for 10 days post infection. Kaplan-Meyer survival curves were made based on the observation of 12-18 mice/group from two independent experiments. The P-values were calculated using the log-rank test (ΔTYAtg7+/+ vs ΔTYAtg7-/-, P = 0.2186; ΔSTYAtg7+/+ vs ΔSTYAtg7-/-, P=0.0145).
Pseudomonas aeruginosa survives in epithelia by ExoS-mediated inhibition of autophagy and mTOR (2020)
Figure 5

Figure 5. ExoS ADP-ribosyltransferase activity inhibited autophagy and mTOR signaling pathway.

(A,F) A549 cells were infected for 4 hours with ExoS-deficient P. aeruginosa (ΔS), ExoS containing P. aeruginosa (SWT), P. aeruginosa containing ExoS with loss-of-function mutations in the GTPase activating domain (SG-A+), in the ADP-ribosylation domain (SG+A-), or in both domains (SG-A-). Cell lysates were evaluated by immunoblotting.

(B) A549 cells were transfected, for 24hours, with a plasmid expressing empty vector (EV), WT GFP-ExoS, a GFP-ExoS mutant for ADPRT domain (SG+A-) or a GFP-ExoS mutant deficient in both GAP and ADPRT domains (SG-A-). Cell lysates were evaluated by immunoblotting.

(C) A549 cells were treated for 24 hours with 100 µM of the ADPRT activity inhibitor MIBG and then left uninfected (NI) or infected with P. aeruginosa containing ExoS with ADPRT activity only (SG-A+). Cell lysates were evaluated by immunoblotting.

(D,E) A549 cells were infected for 4 hours with WT or P. aeruginosa mutants (ΔpscD, ΔSTY, ΔS and ΔTY). Cell lysates were evaluated by immunoblotting.
Pseudomonas aeruginosa survives in epithelia by ExoS-mediated inhibition of autophagy and mTOR (2020)
Figure 7

Figure 7. ADP-ribosyltransferase activity of ExoS was required for its effect on omegasome formation.

(A) A549 cells were transfected, for 48 hours, with a mCherry-DFCP1 plasmid and then infected, for 4 hours, with P. aeruginosa (WT, ΔSTY, ΔS, ΔTY, SG-A-, SG-A+ or SG+A-) and the mCherry puncta number per cell was analyzed by fluorescence microscopy. Representative immunofluorescence images were showed, and scale bars represent 10 µm.

(B) Quantitative analysis of mCherry-DFCP1 puncta by visually count more than 100 cells for each sample. Each bar represents mean value of from three independent experiments and error bars represent standard deviation. The significance of differences between assays was determined using ANOVA with Dunnett multiple-comparison posttest, NS: not significant; *p ≤ 0.05; **p ≤ 0.01.
Pseudomonas aeruginosa survives in epithelia by ExoS-mediated inhibition of autophagy and mTOR (2020)
Figure 8

Figure 8. ExoS ADP-ribosyltransferase activity inhibited Vps34 kinase activity.

(A, C) A549 cells, transfected for 48 hours with vector encoding Flag-Atg14L, were infected with P. aeruginosa (WT, ΔSTY, ΔS, ΔTY, SG-A-, SG-A+ or SG+A-). Flag-Atg14L-Vps34 complex was immunoprecipitated (IP) by Flag-antibody magnetic beads and evaluated by immunoblotting.

(B, D) The IP complexes were subjected to in vitro kinase assay using phosphatidylinositol as substrate. The products radiolabeled PI3P were separated by TLC and detected by autoradiography. Data are mean ± SD of three independent experiments. The significance of differences between different assays was determined using One-way ANOVA analysis followed Dunn's multiple-comparison posttest, NS: not significant; *p ≤ 0.05.

EV Figure Legends
Tubulin polyglutamylation differentially regulates microtubule-interacting proteins (2022)
Figure 1

Figure 1. Purification of brain tubulin with altered posttranslational modifications.

A) Schematic representation of the tubulin PTMs acetylation and polyglutamylation, both highly enriched on brain tubulin. The acetylation of α-tubulin at lysine-40 (K40) is catalysed by ATAT1 and is detected by the 6-11B-1 antibody (Appendix Table S2). Polyglutamylation takes place within the C-terminal tubulin tails of both α- and β-tubulin, and is catalysed by TTLL1 and TTLL7, respectively. Glutamate chains of longer than three residues, predominantly found on α-tubulin, are specifically detected by polyE antibody, while a β-tubulin-specific monoglutamylation at amino acid residue 435 (β2-tubulin) is detected by β-monoE antibody (Bodakuntla et al, 2021) (Appendix Table S2).

B) Overview of the PTM patterns of tubulin purified from knockout mouse models used in this study (Bodakuntla et al, 2021).

C) Schematic overview of the tubulin purification pipeline from mouse brains using three cycles of temperature-induced polymerisation-depolymerisation, including a polymerisation step in high molarity buffer to remove microtubule-associated proteins.

D) SDS-PAGE analysis of purified tubulin from brains of all mouse models used in the current study, stained with Coomassie Brilliant-Blue. Alpha- and β-tubulin are separated using a specific SDS-PAGE protocol (Souphron et al, 2019).

E) Immunoblot analysis of all purified tubulin variants. The molecular weight in (D) and (E) is indicated in kDa.
Tubulin polyglutamylation differentially regulates microtubule-interacting proteins (2022)
Figure 2

Figure 2. Polyglutamylation controls the affinity of Tau protein to microtubules.

A) Schematic representation of the TIRF microscopy assay setup. Label-free GMPCPP-stabilised microtubules, polymerised from wild-type and knockout-mouse brain tubulin, are attached to a glass coverslip chamber sequentially and imaged in IRM mode. Their identity is retained by their positions on the coverslip (Steps 1 and 2). Next, 70 nM purified Tau-meGFP is added to the chamber and its microtubule binding recorded in TIRF microscopy mode (Step 3). The fluorescence intensity of Tau-meGFP on each microtubule in the chamber is measured in the subsequent affinity analysis (Step 4).

B) Representative IRM and TIRF microscopy images showing microtubule positions and Tau-meGFP intensities for different PTM variants (grey: wild type; blue: Ttll1-/-; yellow: Ttll7-/-; green: Ttll1-/-Ttll7-/-; pink: Atat1‑/‑). In the last column, false-coloured TIRF images highlight differences of Tau-meGFP intensities on different types of microtubules (Movie EV1). Scale bars 5 µm.

C) Quantification of integrated intensity of Tau-meGFP on GMPCPP microtubules in three independent sets of experiments performed with independently purified tubulin samples. Single assays (Fig EV2A) of one experiment were combined after normalisation to the wild-type microtubule values (A.U. = arbitrary units). Each data point represents the normalised fluorescence intensity value of one microtubule. Mean values and standard deviation shown for each scatter plot. Student's t-test; p-values displayed.

D) Summary quantification the three independent experiments shown in (C). Each point is a normalised mean of the triplicates, error bars represent standard deviation. Student's t-test; p-values displayed.

E) Saturation curve of increasing concentration Tau-mCherry binding to GMPCPP wild-type and Ttll1-/-Ttll7-/- microtubules. Each data point is shown as mean ± standard deviation of the raw integrated intensity values (A.U. ×104) of three experiments with independently purified tubulin samples (Fig EV2D). Dissociation constants Kd and R2 coefficient from non-linear fits of each microtubule type are indicated below the plot.

F) Tau-envelope formation on Taxol-stabilised wild-type and Ttll1-/-Ttll7-/- microtubules (grey and green arrowheads, respectively) 5 and 120 s after addition of 40 nM Tau-mCherry to the TIRF microscopy flow-chamber (Movie EV2). Scale bars 5 µm.

G) Analysis of percentage total microtubule length in the flow-chamber covered by Tau envelopes. Pooled data points from three experiments (each data point represents tau coverage of microtubules within one field of view) with medians and interquartile ranges represented. Mann-Whitney test, p-values displayed.
Tubulin polyglutamylation differentially regulates microtubule-interacting proteins (2022)
Figure 4

Figure 4. Selective impact of polyglutamylation patterns on kinesin-1 motility

A) Schematic representation of the single-molecule kinesin-1 motility assay. Taxol-stabilised microtubules of two different types are subsequently attached in the flow-chamber (Steps 1, 2), 0.2 nM Kif5b-EGFP are added, the motility of single kinesin motors recorded in TIRF microscopy (Step 3) and subsequently analysed (Step 4).

B) Kymographs showing single Kif5B-EGFP motor molecules moving on one single microtubule of known PTM state (Movie EV4). Horizontal scale bars 2 µm; vertical scale bars 5 s.

C) Scatter plots showing the average velocities (µm/s) exhibited by single Kif5b-EGFP molecules on the different microtubule types (each data point represents one Kif5b-EGFP molecule). Data pooled from 29 independent experiments (Appendix Fig S2). Medians with interquartile ranges shown, Mann-Whitney test, p-values displayed.

D,E) Survival probability (Kaplan-Meier plots, taking varying microtubule lengths into account; see Methods) of the interaction time (D) and run length (E) of Kif5b-EGFP molecules on different microtubule types. Data in (C,D,E) were pooled from individual experiments (Appendix Fig S2) without normalisation. The 50% probability values are show in the plots. Statistical analysis displayed in the tables below: hazard ratios with 95% confidence intervals pairing all data from different microtubule types (*pairs differing with significance level p<0.05).
A pH- and ionic strength-dependent conformational change in the neck region regulates DNGR-1 function in dendritic cells (2016)
Figure 1

Figure 1. DNGR-1 is a glycosylated disulphide-bonded dimer

(A) Western blot analysis of supernatants containing long and short mouse and human DNGR-1 ECD proteins under reducing and non-reducing conditions. (B) Western blot analysis of supernatants after protein production of WT and C94S mutant long mouse DNGR-1 ECD proteins under reducing and non-reducing conditions. C94S mutant-containing supernatant was 200x concentrated before analysis. (C) Western blot analysis of supernatants containing the indicated DNGR-1 ECD proteins after treatment with PNGase F overnight. HRP-conjugated anti-FLAG antibody was used for detection of all proteins. Numbers on the side of blots indicate positions of molecular weight markers.
A pH- and ionic strength-dependent conformational change in the neck region regulates DNGR-1 function in dendritic cells (2016)
Figure 2

Figure 2. DNGR-1 can form reduction resistant dimers

(A) 0.25 μg of purified long mouse DNGR-1 ECD was diluted into 10 mM Tris buffer of the indicated pH (left panel) or into 10 mM Tris pH 7.4 buffer supplemented with increasing amounts of NaCl (1 – 250 mM; right panel), and reduction sensitivity of DNGR-1 was assessed by reducing SDS-PAGE and Western blot. (B) 0.25 μg of purified DNGR-1 long mouse ECD was diluted into buffers of different pH and ionic strength, and its reduction sensitivity was assessed by reducing SDS-PAGE and Western blot. The intensity of bands corresponding to dimer and monomer was determined densitometrically, and the ratio was plotted as a function of buffer ionic strength and pH. (C) 0.25 μg of purified long mouse DNGR-1 ECD was diluted into PBS or 10 mM MES pH 6.1 buffer, and its reduction sensitivity was tested under mildly (Laemmli buffer) or strongly denaturing (8 M urea) conditions by reducing SDS-PAGE and Western blot. (D) 0.25 μg of purified long mouse DNGR-1 ECD was diluted into PBS or 10 mM MES pH 6.1 buffers, and its reduction sensitivity after different lengths of heat-denaturation in Laemmli buffer was tested by reducing SDS-PAGE and Western blot. HRP-conjugated anti-FLAG antibody was used for detection of all proteins. Numbers on the side of blots indicate positions of molecular weight markers. Reduction-resistant dimers are indicated with an * and reduction-sensitive protein with a +.
A pH- and ionic strength-dependent conformational change in the neck region regulates DNGR-1 function in dendritic cells (2016)
Figure 3

Figure 3. Formation of reduction resistant dimers is reversible and conserved between DNGR-1 isoforms

(A) 1 μg of purified long mouse DNGR-1 ECD was transferred into 10mM MES pH 6.1 and 0.5 μg into PBS. Half of the MES sample and the whole PBS sample were dialyzed against 2 l of PBS overnight at 4°C. After dialysis, all samples were prepared for reducing SDS-PAGE and Western blot. (B) Supernatants after production of indicated proteins in 293F cells were harvested and diluted into indicated buffers at a 2:3 ratio and analyzed using reducing SDS-PAGE and Western blot. HRP-conjugated anti-FLAG antibody was used for detection of all proteins. Numbers on the side of blots indicate positions of molecular weight markers. Reduction-resistant dimers are indicated with an * and reduction-sensitive protein with a +.
A pH- and ionic strength-dependent conformational change in the neck region regulates DNGR-1 function in dendritic cells (2016)
Figure 4

Figure 4. The neck of DNGR-1 is sufficient for formation of reduction resistant dimers

(A) Schematic representation of DNGR-1, Dectin-1 and the chimeric protein consisting of the neck region of DNGR-1 and CTLD of Dectin-1. (B) Supernatants containing ECD of long mouse DNGR-1, Dectin-1 and the chimeric receptor were prepared for SDS-PAGE and Western blot under non-reducing conditions. (C) Supernatants containing indicated proteins were diluted into PBS or pH 6.1 MES buffer and analyzed using SDS-PAGE and Western blot under reducing conditions. HRP-conjugated anti-FLAG antibody was used for detection of all proteins. Numbers on the side of blots indicate positions of molecular weight markers. Reduction-resistant dimers are indicated with an * and reduction sensitive protein with a +.
A pH- and ionic strength-dependent conformational change in the neck region regulates DNGR-1 function in dendritic cells (2016)
Figure 5

Figure 5. A change in the tertiary structure is responsible for the formation of reduction-resistant dimers

(A) Long mouse and human DNGR-1 ECD proteins were diluted in PBS or 10 mM MES pH 6.1 buffer and 20 independent far-UV spectra were acquired for each condition (red and black lines depict the composite curve for each condition) (B) Long mouse DNGR-1 ECD was diluted in PBS or 10 mM MES pH 6.1 buffer and 20 independent near-UV spectra were acquired for each condition (red and black lines depict the composite curve for each condition and the blue line shows the difference between the two). (C) Schematic representation of the suggested conformational states of DNGR-1.