EMBO/soda-nel huggingface dataset:
CARD8 inflammasome signaling in human T cells (2020)
Figure 1

Figure 1 - Dipeptidyl-peptidase inhibition triggers a lytic cell death in human primary T cells

(A and B) FACS-sorted T cell populations and Monocyte-derived Macrophages were treated with the indicated stimuli: Nigericin (Nig. 4h), NeedleTox (4h), Val-boroPro (VbP 22h), ABT737/S63845 (A/S 22h). When indicated, cells were primed with LPS for 2 hours prior to stimulation. When indicated, MCC950 was added to the media 30 minutes prior to the addition of Nigericin. LDH-activity (A) and IL-1β and IL-18 concentration (B and C, respectively) in the supernatant were determined by LDH cytotoxicity assay and ELISA respectively. Individual data points ± SEM from three independent donors are shown. Statistics indicate significance by two-way ANOVA: ***p ≤ 0.001; **p ≤ 0.01; *p ≤ 0.05; ns, not significant. P-values were corrected for multiple comparisons (Dunnett).
CARD8 inflammasome signaling in human T cells (2020)
Figure 2

Figure 2 - VbP-treated T cells display hallmarks of pyroptosis

(A) Macrophages and CD4 T cells from the same donor were subjected to the indicated treatments and morphologic changes as well as PI-uptake were monitored by live-cell imaging microscopy using a 25x objective. Representative images from indicated time points are shown. Cyan color coding is used for the fluorescent PI-signal. One donor out of two is shown.

(B) Representative images were acquired with a 63x objective at the end of the experiment shown in (A) at 16 hours. One donor out of two is shown. Scale bars: 25 µm.

(C) CD4 T cells were treated with VbP or ABT737/S63845. Lysate and supernatant samples were collected at the indicated time points. Samples were analyzed by immunoblotting. αSS and αLS indicate the use of a small subunit or large subunit-specific caspase-1 antibody respectively. (Lys = lysate, SN = supernatant, FL = full length). One representative experiment out of three is shown.
CARD8 inflammasome signaling in human T cells (2020)
Figure 4

Figure 4 - DPP9 inhibition operates upstream of CARD8 engagement

(A) Schematic of the experimental setup

(B) MiSeq analysis of the indicated gene targeting approaches from samples collected at the indicated time points post editing. DPP9 and DPP8 were targeted with one gRNA/target gene, CARD8 was targeted with two gRNAs. On the day of nucleofection, T cells were activated with human T activator CD3/CD28 beads and expanded in the presence of IL-2. On day 10, cells were switched to IL-7/IL-15 containing medium.

(C) Fraction of wildtype and in-frame reads are depicted. One representative experiment out of three is shown.
CARD8 inflammasome signaling in human T cells (2020)
Figure 5

Figure 5 - TCR stimulation renders T cells resistant to pyroptosis

(A) CD4 T cells were activated with human T activator CD3/CD28 beads (bead:cell ratio 1:5) for 4 days. Controls were kept resting in the presence of IL-7 and IL-15. On day 4 cells were stimulated as indicated for 18 hours and cytotoxicity was assessed by LDH-activity in the supernatant. Individual data points ± SEM from four independent donors. Statistics indicate significance by two-way ANOVA: ***p ≤ 0.001; ns, not significant. P-values were corrected for multiple comparisons (Sidak).

(B) Immunoblotting of CD4 T cell lysates treated as in (A) for 3 to 4 days. * indicates an unspecific band.

(C) Quantification of immunoblots shown in (B) and additional donors (n= 9 for all but GSDMD, GSDMD n=8, for one donor quantification of the GSDMD band was considered unreliable due to uneven substrate distribution). Individual data points ± SEM are shown. Statistics indicate significance by one-way ANOVA of log2-transformed fold change values of band intensities (activated/non-activated T cells) normalized to β-Actin: ***p ≤ 0.001; *p ≤ 0.05; ns, not significant. P-values were corrected for multiple comparisons (Sidak). All band intensity ratios were tested against β-Actin. CARD8 full length was additionally tested against CARD8 cleaved.

Expanded View Figure legends
Generation and deposition of Aβ43 by the virtually inactive presenilin-1 L435F mutant contradicts the presenilin loss of function hypothesis of Alzheimer ́s disease (2016)
Figure 1

Figure 1. The PS1 L435F FAD mutant strongly impairs APP processing while generating Aβ43

Pooled clones (A–D) and single cell clones (E–H) of HEK293/sw cells untransfected or stably transfected with the indicated WT and mutant PS1 constructs were analyzed for γ-secretase expression and APP processing.

(A) PS1, PS2 and NCT were analyzed in cell lysates by immunoblotting using antibodies PS1N (PS1), BI-HF5C (PS2) and N1660 (NCT), respectively.

(B) Full length APP and APP CTFs were analyzed by immunoblotting using antibody 6687. (C) Conditioned media were analyzed for secreted APPs by immunoblottting using antibody 22C11 and for total Aβ by combined immunoprecipitation/immunoblotting using antibodies 3552/2D8.

(D) Conditioned media were analyzed for individual Aβ species on Tris-Bicine urea SDS-PAGE by combined immunoprecipitation/immunoblotting of Aβ using antibodies 3552/2D8. Pooled clones of HEK293/sw cells stably transfected with PS1 L166P were used as reference.Note that more sample was loaded for the PS1 L435F mutant to facilitate analysis.

(E) PS1 expression levels were analyzed in cell lysates of HEK293/sw cells stably expressing PS1 WT or PS1 L435F by immunoblotting using antibodies PS1NT and 5E12, respectively.

(F) Conditioned media were analyzed by ELISA specific for Aβ40, Aβ42 and Aβ43. Data represent mean ± s.e.m. (n=6). Absolute levels and Aβ ratios are shown.

(G) Secreted Aβ was analyzed as in (D). To verify individual Aβ species, Aβ standards were co-migrated.

(H) Total Aβ in conditioned media was analyzed by MALDI-TOF MS following immunoprecipitation with antibody 4G8. Observed (Aβ42, 4513.6; Aβ43, 4615.3) and predicted molecular masses (Aβ42, 4514.1; Aβ43, 4615.2) were in good agreement.
Generation and deposition of Aβ43 by the virtually inactive presenilin-1 L435F mutant contradicts the presenilin loss of function hypothesis of Alzheimer ́s disease (2016)
Figure 2

Figure 2. The PS1 L435F FAD mutant generates Aβ43 as the predominant long Aβ species independent of PS1 WT

(A) PS1/2-/- MEF cells stably transduced with PS1 WT or PS1 L435F were analyzed for PS1 expression and APP processing by immunoblotting as in Fig 1A. Antibody 5E12 was used for the detection of the PS1 CTF.

(B) Stably transduced PS1/2-/- MEF cells of A were transiently transfected with APPsw-6myc and conditioned media were analyzed for Aβ species by Tris-Bicine urea SDS-PAGE. Individual Aβ species were verified by co-migration with Aβ standards (left panel) and quantified (right panel). Data represent mean ± s.e.m. of n=3 independently performed transfections. Note that more sample was loaded for the PS1 L435F mutant to facilitate immunoblot analysis.

(C) Secreted Aβ from cells in B were quantified by ELISA. Data represent mean ± s.e.m. of n=5 (PS1 WT) or 6 (PS1 L435F) independently performed transfections.
Generation and deposition of Aβ43 by the virtually inactive presenilin-1 L435F mutant contradicts the presenilin loss of function hypothesis of Alzheimer ́s disease (2016)
Figure 3

Figure 3. Deposition of Aβ isoforms in the frontal cortex of AD cases with and without PS1 L435F mutation.

Immunohistochemical detection of Aβ40 (left column), Aβ42 (medium column) and Aβ43 (right column) in consecutive frontal cortex paraffin sections of two FAD cases with PS1 L435F mutation (cases #1 and #2, 1st and 2nd rows), another FAD case with different PS1 mutation (case #3, 3rd row) and one SAD case (case #4, forth row). In both PS1 L435F mutation cases the numerous Aβ plaques mainly contain Aβ42 and Aβ43 but less Aβ40. This is in contrast to Aβ plaques of the control FAD and SAD cases, in which solely Aβ42 predominates and Aβ43 is sparse; even levels of Aβ40 seem to be lower than in the PS1 L435F cases. Note that in both PS1 L435F cases several plaques are larger than those seen in cases #3 and #4, representing cotton wool plaques. Scale bar = 500 µm. Magnification is identical in all pictures.
Local actin nucleation tunes centrosomal microtubule nucleation during passage through mitosis (2019)
Figure 1

Figure 1 - Dynamics of actin and microtubule networks during mitotic exit

A. Maximum projection (2 z-slices) of HeLa cells immunostained for F-actin (Phalloidin), Tubulin, Pericentrin and DAPI at metaphase and early anaphase showing actin accumulation around centrosomes in early anaphase. Scale bar- 10µm

B. Quantification of F-actin (Phalloidin) intensity around pericentrin positive centrosomes in HeLa cells immunostained as in (A), showing the increase in F-actin around centrosomes at early anaphase. Mean actin metaphase = 1 ± 0.03996, n=92, mean actin anaphase = 1.875 ± 0.08895, n=121, Student t-test, ****p<0.0001;

C. Time-lapse sequence from a representative Hela cell expressing Lifeact-GFP transiting from metaphase to anaphase, showing accumulation of actin in the presumptive centrosomal region, red arrows. Images represent 4z-projection. Scale bar-10µm.

D. Time-lapse sequence of a representative HeLa cell expressing GFP-alpha-tubulin and RFP-Lifeact transiting from metaphase to anaphase showing the changes in actin and microtubules. Images represent 4 z-projection of movies taken every 1min. Bottom - higher magnification view of the centrosomes C1 and C2 from time-lapse above showing a transient increase in actin around the centrosomes in early anaphase. Scale bar = 10µm, 1µm in zoom. T=0 is one frame before anaphase onset. Dotted circle shows centrosome position.

E. Integrated fluorescence intensity of GFP-alpha-tubulin and RFP-Lifeact around the centrosomes for a population of cells exiting mitosis, showing actin accumulation around the centrosomes and a simultaneous decrease in tubulin over time. N=30 centrosomes (15 cells). T=0 is one frame before anaphase onset. Graph shows mean +/- SD, values were normalized to intensity at T=0.

F. Quantification and comparison of actin (RFP-Lifeact) and tubulin (a-tubulin-GFP) fluorescence intensity around the centrosomes at metaphase (-4 to -1min), early anaphase (2-5min) and late anaphase (7-10min) for a population of cells. Mean actin intensity at Metaphase =1.002 ± 0.0239, n=97, Early Anaphase = 1.023 ± 0.03047, n=101, Late anaphase = 0.9703 ± 0.05004, n=98. Tubulin intensity at Metaphase = 1.008 ± 0.03356, n=106, Early anaphase = 0.9748 ± 0.04124, n=116, Late anaphase = 0.9276 ± 0.05382, n=101, One-way ANOVA.
Local actin nucleation tunes centrosomal microtubule nucleation during passage through mitosis (2019)
Figure 2

Figure 2 - Actin dynamics during forced mitotic exit

A. Stills from time-lapse of HeLa cells expressing GFP-alpha-tubulin and RFP-lifeact arrested at prometaphase with STLC (t=0) and forced to exit mitosis with Cdk1 inhibition (Ro3306) imaged every 90 seconds. Scale bar = 10µm and for zoom = 4µm. n = 21 cells from 4 independent experiments.

B. Quantification of actin around centrosomes when cells are forced to exit mitosis using Cdk1 inhibition as in (A), showing a similar accumulation of actin as observed in bipolar divisions. Graph shows mean actin accumulation normalized to t=0, errors bars indicate standard deviation.

C. HeLa cells expressing GFP-centrin1 arrested at prometaphase or forced mitotic exit (Ro3306 5') and stained with phalloidin. Scale bar- 5µm and for zoom-2µm.

D. The level of actin around the centrosome in (C) was quantified and normalised relative to metaphase and shows an increase during forced mitotic exit. STLC arrest 1 ± 0.01317, n=210, STLC+Ro-5mins=1.251 ± 0.02441, n=144, Unpaired t-test with Welch correction, ****p<0.0001.

E. Quantification of tubulin intensity around centrosomes during prometaphase arrested cells or cells forced to exit with Cdk1 inhibitor. Tubulin intensity decreses during forced mitotic exit. STLC-DMSO- 1 ± 0.02407, n=180, STLC-RO-3306- 0.6516 ± 0.02337, n=136. P<0.0001, Welch's t test.
Local actin nucleation tunes centrosomal microtubule nucleation during passage through mitosis (2019)
Figure 3

Figure 3 - Arp2/3 dependent actin accumulation at the centrosome.

A. Maximum projection (2-z slices) view of Hela cells pre-treated with DMSO and 0.2mM CK666 for 15 mins during their mitotic exit showing that treatment with CK666 leads to reduced accumulation of actin around the centrosomes during anaphase. Scale bar-10µm.

B. Quantification of actin around centrosomes for cells treated with DMSO or 0.2mM CK666, showing the reduction in actin accumulation around the centrosomes following CK666 treatment. DMSO-metaphase-1±0.4325, n=54, CK666-metaphase-0.936±0.4604, n=43, DMSO-anaphase-1.8±0.9736, n=76, CK666-anaphase-1.087±0.4597, n=73; one-way ANOVA, p<0.0001. Date pooled from 3 independent experiments.

C. Z-projection of HeLa cells expressing GFP-centrin1 pre-treated with DMSO or 0.2mM CK666 during prometaphase arrest and forced mitotic exit and stained with phalloidin for F-actin.

D. Quantification of the level of actin around the centrosome from (C), which shows the reduction in actin accumulation around centrosomes following CK666 pre-treatment. DMSO-STLC= 1 ± 0.02768, n=99, CK666 STLC= 0.776 ± 0.02186, n=87, DMSO-RO-3306= 1.339 ± 0.03048, n=127, CK666-RO-3306= 0.7699 ± 0.02246, n=70, one-way ANOVA, p<0.0001.

E. Quantification of tubulin around centrosomes for cells treated with DMSO or 0.2mM CK666, showing the failure to reduce tubulin density around centrosomes following CK666 treatment during bipolar exit. DMSO-metaphase-1±0.1968, n=54, DMSO-anaphase-0.867±0.2345, n=78, CK666-metaphase-0.7995±0.1275, n=43, CK666-anaphase-0.7407±0.1859, n=74; one-way ANOVA, p<0.0001. Data pooled from 3 independent experiments. Error bars indicated standard deviation.

F. Quantification of tubulin around centrosomes for cells treated with DMSO or 0.2mM CK666, during monopolar exit, showing the failure to reduce tubulin density around centrosomes following CK666 treatment. Data from three independent experiments. Prometaphase arrest (p.a.) 1±0.34 , n=27; forced mitotic exit (f.m.e) 0.78±0.30, n=26; p.a. plus CK666 0.49±0.34, n=30 ; f.m.e plus CK666 0.92±0.37, n=30. One-way ANOVA with Sidak's multiple comparisons test.
Local actin nucleation tunes centrosomal microtubule nucleation during passage through mitosis (2019)
Figure 4

Figure 4 - Arp2/3 mediated actin nucleation around centrosomes in vitro

A. Z-projection of HeLa cells expressing GFP-centrin1 immunostained for p34-Arc (sub-domain Arp2/3) following prometaphase arrest and forced mitotic exit for 5min. N=3 independent experiments. Scale bar = 10µm and for zoom 2µm.

B. Levels of p34-Arc around the centrosome (3um area) were quantified relative to metaphase and increase during forced mitotic exit. STLC arrested = 1 ± 0.01576, n=153, STLC+RO-3306= 1.268 ± 0.01748, n=191, ****p<0.0001, Unpaired t-test with Welch correction. Error bars represent standard deviation.

C. Z-projection of centrosomes isolated from cells expressing GFP-centrin1 during prometaphase arrest and forced exit, which were immunostained with p34-Arc. N=2 independent experiments. Scale bar = 2µm.

D. Quantification of p34-Arc levels around the centrosome in (C), relative to metaphase, showing its increase during forced exit. STLC arrested = 1 ± 0.00779, n=93, STLC+RO-3306= 1.249 ± 0.02446, n=134, ****p<0.0001, Unpaired t-test with Welch correction. Error bars represent standard deviation.

E. Time-lapse of in vitro assay on centrosomes isolated from prometaphase arrested and cells forced to exit mitosis showing centrosomal actin nucleation over time. Scale bar = 10µm

F. Quantification of actin nucleation from isolated centrosomes as in (E) over an area of 2 μm, (white circle), showing an increase in actin nucleation around centrosomes isolated from cells undergoing forced mitotic exit. N=2 isolations, 5 independent experiments, n=16 STLC and n=19 STLC RO.

G. Color switch experiment using green- and red-labelled actin. Red actin was used first, followed by Capping protein, then green-labelled actin. The accumulation of green in the centre indicates that actin is nucleated at the centrosome. Scale bar = 10µm.

H. Quantification of (G) over the time. N=2 isolations, 2 independent experiments, n=19 STLC and n=14 STLC RO. Error bars represent standard deviation.

I. Time-lapse showing cytoplasmic actin filament formation at centrosomes isolated from cells undergoing forced exit when pre-treated with DMSO or CK666. Scale bar = 10µm.

J. Quantification of (I), showing that centrosomal actin nucleation fails when centrosomes isolated from cells undergoing exit are pre-treated with CK666. N=2 isolations, 2 independent experiments, n = 17 DMSO, 26 Ro CK666 24 STLC DMSO, 23 STLC CK666. Error bars represent standard deviation.
Local actin nucleation tunes centrosomal microtubule nucleation during passage through mitosis (2019)
Figure 5

Figure 5 - Localization and post-translational modifications of WASH1 during mitosis

Phos-tag Western blot of MCF7-Her2-18 cells synchronised into distinct phases of the cell cycle showing the phospho-shift in WASH1 during mitosis.

Blue native PAGE Western blot showing shift in the size of WASH1 protein complex isolated from STLC treated prometaphase arrested cells when compared to cells forced to exit mitosis following Ro3306 treatment.
Local actin nucleation tunes centrosomal microtubule nucleation during passage through mitosis (2019)
Figure 6

Figure 6 - Anaphase centrosomal actin aster formation depends on the Arp2/3 activator WASH1

A. Time-lapse of HeLa cells expressing GFP-alpha tubulin and RFP-lifeact treated with control siRNA or siRNA against WASH1 for 48 hours arrested at prometaphase and forced to exit mitosis with Ro3306 addition (t=0). Scale bar = 5µm.

B. Quantification of actin around the centrosome in (A), showing the failure to accumulate actin around the centrosome in siWASH. N = 4 experiments. Error bars represent standard deviation.

C. Western blots of lysates from cells treated with siRNA control and siRNA against WASH1 probed with WASH1 antibody, showing the reduction in WASH levels following treatment of cells with siRNA against WASH. Scale bar = 5µm and 2µm in zoom.

D. Z-projection of HeLa cells expressing GFP-centrin1 treated with control siRNA or siRNA against WASH, arrested at prometaphase or forced to exit mitosis and immunostained with phalloidin. Scale bar-5µm

E. Quantification of actin around the centrosome in (F), showing the failure to accumulated actin around the centrosome during forced exit in cells treated with siRNA against WASH. siControl-STLC= 1 ± 0.0189, n=165, siWASH-STLC=0.9138 ± 0.0298, n=118, siControl-STLC+RO-3306=1.911 ± 0.06221, n=147, siWASH-STLC+RO-3306=1.103 ± 0.0301, n=140. Error bars represent standard deviation. P<0.0001, one-way ANOVA.

F. Time-lapse from in vitro assay with centrosomes isolated from cells undergoing forced exit were pre-treated with either no antibody or anti-WASH1 antibody for 1 hour. Scale bar- 10µm

G. Quantification of in vitro assay from (F), showing the reduction in actin nucleation around centrosomes when they are pre-treated with anti-WASH1. Error bars represent standard deviation. 2 independent experiments.

Figure Legend for EV figures-
Systems approach reveals photosensitivity and PER2 level as determinants of clock-modulator efficacy (2019)
Figure 4

Figure 4. CK1i induces a larger phase delay as PER2 levels increase depending on the molecular cause of ASPD and external lighting conditions.

A The simulated period of ASPD models is shorter than that of the WT model in DD. Thus, they have a ~4 h more advanced phase than the WT model in LD 12:12 (inset). PER2 protein concentrations in each mutant are normalized to the maximum PER2 concentration of the WT model (dashed gray line; inset).

B The phase delays induced by a single daily dosing of PF-670 were simulated. The dosing regimens inducing ~4 h phase delays in the WT model led to much larger phase delays in all ASPD models with large inter- and intra-variations (blue circles). When the dosing times are advanced by 4 h reflecting the advanced phase of the ASPD models, the phase delays become closer to but still larger than the ~4 h phase delays (red squares).

C As the average PER2 level is higher in the ASPD models (A), PF-670 induces a larger phase delay. Here, the phase shift is the average of the phase delays represented as the red squares in (B). The line represents the least-square fitting line. r and P denote the Pearson's correlation coefficient and P-value of Pearson's correlation test, respectively. α denotes the slope of the least-square fitting line.

D The ASPD models simulate the larger PF-670-induced phase delay in LD 8:16 than in LD 16:8 due to the higher PER2 level in LD 8:16 than in LD 16:8 when dosing occurs (Fig EV5G).

E Consistently, higher experimentally measured PER2 levels in the SCN (Amir et al, 2004) at the dosing time leads to a larger PF-670-induced phase delay (Badura et al, 2007), which was also captured by the NHP model when 20 mpk dosing was used.
Slit-Robo Signalling Establishes a Sphingosine-1-Phosphate Gradient to Polarise Fin Mesenchyme (2022)
Figure 4

Figure 4: Mesenchymal cells of both mutants show abnormal morphology and loss of polarity

A-C: Confocal projections of the 40hpf tails of WT (A), slit3sq49 (B) and s1pr2te273 (C), crossed to sqet37Et, labelling the fin mesenchyme in eGFP. Insets show transverse orthogonal slice at the indicated location. The mutant mesenchymal cells are attached to the inner wall of the blister as indicated by arrowheads. Scale bar indicates 20µm.

D-F: Changes in mesenchyme circularity (D), eccentricity, and length (E) as the cell migrates away from the paraxial mesoderm, between 30hpf-40hpf. Three embryos were tracked for each condition, and 33 cells of WT, 33 cells of slit3sq49 and 35 cells of s1pr2te273 were analysed.

G-I: WT cells (G) close to the apex have an elongated and polarised appearance whilst both mutants are unpolarised and have a disc like appearance (H,I, centre and right panels).

J-L: Tracks of cells from WT (J) slit3-/- (K) and s1pr2-/- (L) embryos over 60 minutes duration. Mutants display a lack of directionality and reduced displacement over a short range. Tracks are normalized to a common start point, 23 cells of WT, 22 cells of slit3sq49 and 21 cells of s1pr2te273 from 3 to 4 embryos were tracked.

M-O: Schematic describing the measurement of a cell's approach angle to the nearest point on the AER (M). Merged, immunofluorescent images of WT (top), slit3sq49 (centre) and s1pr2te273 (below) mesenchymal cells in sqet37Et background stained for EGFP (green), Gamma tubulin (red) and DAPI (blue). White lines run from the centre of nucleus to the nearest point on the AER, through the MTOC (N). Graph depicting the approach angles to AER of leading (Tier 1), following (Tier 2) and trailing (Tier 3) cells of WT, slit3sq49 and s1pr2te273 embryos (O). A minimum of 30 cells were measured for each tier of each genotype. *p<0.05; **p<0.005; ***p<0.001; ANOVA with Fisher's post-test.

Data information: Scale Bars: 50µm (A), 10µm (G).