EMBO/soda-nel huggingface dataset:
DASH/Dam1 complex mutants stabilize ploidy in histone-humanized yeast by weakening kinetochore-microtubule attachments (2022)
Figure 7

Figure 7. DAD1E50D disrupts DASH complex oligomerization and weakens microtubule attachments

(a) Structure of the 17-member DASH/Dam1c ring of C. thermophilum around a microtubule lattice is shown (6CFZ and 5SYF, respectively). The ring, with a 390 Å interior circumference, encircles a single microtubule with a width of 250 Å (dashed line). Individual protomers oligomerize through interactions of two conserved interfaces, with the DAD1E50 and DAM1N80 residues highlighted in the zoomed in region of interface II.

(b) Analysis of DASH complex rings around microtubules. Top, example images are shown for the three cases measured (bare microtubules, WT DASH, and DAD1E50D DASH). The bare microtubule lattice was measured from the same micrographs with DASH complex present, where sections that showed no DASH complex were measured. Below, the z-score normalized pixel intensities for the example EM micrographs. Note the increased intensity outside the range of the diameter of the microtubule lattice for samples with DASH complex.

(c) Heatmaps show the z-score normalized pixel intensities along a straight edge bisecting the width of the microtubule or microtubule + DASH complex. The number of particles analyzed is shown in parentheses. Above are the average profiles of pixel intensity with 95% CI. Dashed gray line is the average profile of pixel intensity of bare microtubules.

(d) Size exclusion chromatography of the indicated DASH complexes.

(e) Schematic of the optical trap experiment with wildtype and mutant DAD1E50D DASH complex on microtubules.

(f) Rupture force assay from experiments at three concentrations of DASH complex incubated with the beads. Briefly, 3.5 pM of beads were incubated with the indicated concentrations of DASH complex (representing a ratio of DASH complex to beads of 714:1, 1428:1, and 2860:1). These DASH-decorated beads were then washed before suspending in a reaction mix containing free tubulin and 5 nM free DASH complex. Two way ANOVA tests of the mean difference in rupture force are shown with multiple test corrections (false discovery rate). Each dot represents an individual rupture force measurement (n ≥ 21; exact number shown below each box between parentheses), central band represents the median, box extends from the 25th to 75th percentiles, and whiskers represent the minimum and maximum.

(g) Survival curves for each DASH complex are shown at the indicated concentrations. Log-rank tests were performed to test whether the survival curves were significantly different from one another. Error bars represent the standard error and are displayed as the shaded area.
DASH/Dam1 complex mutants stabilize ploidy in histone-humanized yeast by weakening kinetochore-microtubule attachments (2022)
Figure 8

Figure 8. DAD1E50D decreases the efficiency of sporulation and spore viability

(a) Sporulation efficiency after five days in sporulation medium at 25 ˚C. Example micrographs are shown to the left for both wildtype and DAD1E50D SK1 strains. Right, quantification of average sporulation efficiency from all micrographs. Two tailed unpaired t test of the mean difference in sporulation efficiency. Each dot represents the fraction of cells sporulated in a single field of view (technical replicates; n ≥15), central band represents the median, box extends from the 25th to 75th percentiles, and whiskers represent the minimum and maximum.

(b) Spore viability assay. Individual tetrads were micromanipulated on a agar surface and spores were dissected and arrayed. Genotype was determined by PCR genotyping of the DAD1 locus. Quantification from all tetrad dissections performed, total are shown below. The one-tailed probability of observing 51% spore viability in DAD1E50D SK1 over a total of 139 tetrads is p < 0.000001, assuming a spore viability of ~90% from wild type SK1.

(c) Genotyping of spores isolated from tetrads with either four viable spores or two viable spores. Fraction of spores with the indicated allele (gray DAD1WT and pink DAD1E50D) is displayed. The one-tailed binomial test is shown for the probability of observing the frequency of the DAD1E50D allele given equal segregation (DAD1WT, P = 0.46; DAD1E50D, P = 0.0011). Numbers below each bar indicates the total number of spores genotyped.

Expanded View Figure Legends
Structural basis for the recognition of the 2019-nCoV by human ACE2 (2020)
Figure 1

Overall structure of the RBD-ACE2-B0AT1 complex.(A) Cryo-EM map of the RBD-ACE2-B0AT1 complex. Left: Overall reconstruction of the ternary complex at 2.9 Å. Inset: focused refined map of RBD. (B) Overall structure of the RBD-ACE2-B0AT1 complex. The complex is colored by subunits, with the protease domain (PD) and the Collectrin-like domain (CLD) colored cyan and blue in one of the ACE2 protomers, respectively. The glycosylation moieties are shown as sticks.
Structural basis for the recognition of the 2019-nCoV by human ACE2 (2020)
Figure 2

Cryo-EM density of the interface between RBD and ACE2.The density, shown as pink meshes, is contoured at 12 σ.
Transcription factor FOXP2 is a flow-induced regulator of collecting lymphatic vessels (2021)
Figure 1

Figure 1 - Transcriptome analysis of lymphatic vessels identifies FOXP2 as a collecting vessel-specific transcription factor.

A Isolation of dermal EC subtypes from the ear skin of 5 weeks old Prox1-GFP mice using multicolor flow cytometry. Whole-mount immunofluorescence for LYVE1 (on the left) defines three Prox1-GFP+ lymphatic vessels subtypes that are sorted using the indicated gating scheme (on the right): LYVE1low/- collecting vessel (green arrow, col); LYVE1intermed pre-collecting vessel (grey arrow, pre-col); LYVE1high lymphatic capillary (magenta arrow, cap).

B Top 100 genes up-regulated in collecting in comparison to lymphatic capillary LECs. Expression in BECs is shown for comparison. Heatmap color coding shows log2 fold change. Red box indicates LEC-specific/enriched collecting vessel signature genes that include Foxp2 (red arrow).

C Whole-mount immunofluorescence of ear skin, mesentery and flank, showing nuclear FOXP2 staining in collecting lymphatic vessels (arrows, col), but not in LYVE1+ lymphatic capillaries (cap) or blood vessels (bv).

D qRT-PCR analysis of Foxp1-4 in murine ECs freshly isolated from P11 mesentery (n = 6 mice, individual data points shown). Data are presented as mean relative expression (normalized to Gapdh) ± SD. Transcript levels for each transcript are presented relative to levels in LECs. P, Student's t-test. nd, not detected.

Data information: Scale bar: 75 µm (A), 50 µm (C).
Transcription factor FOXP2 is a flow-induced regulator of collecting lymphatic vessels (2021)
Figure 3

Figure 3 - Endothelial specific deletion of Foxp2 leads to defective collecting vessel and valve morphogenesis.

A Genetic constructs and breeding strategies used to generate pan-endothelial Foxp2flox/flox;Tie2-Cre (grey) or Foxp2flox/-;Tie2-Cre (blue) deletion.

B, C Whole-mount immunofluorescence of P7 mesenteries, showing abnormally short integrin-α9+ lymphatic valve leaflets (B) and selection of non-recombined FOXP2+ LECs (C) in Foxp2flox/flox;Tie2-Cre mice.

D Whole-mount immunofluorescence of P7 mesenteries, showing efficient FOXP2 depletion in Foxp2flox/-;Tie2-Cre vessels. Dotted line indicates vessel width.

E Quantification of mesenteric collecting lymphatic vessel width in P7 Foxp2flox/-;Tie2-Cre and littermate controls (n = vessels [mice], as indicated).

F Lymphatic valves in P7 mesenteric vessels of control and Foxp2flox/-;Tie2-Cre mice. A spectrum of defects in the mutants ranging from shortened integrin-α9+FN-EIIIA+ valve leaflets (middle panels) to a complete loss of leaflets (right panels) are shown.

G, H Quantification of the proportion of valves with integrin-α9+ leaflets (G), and leaflet length (H) in P7 Foxp2flox/-;Tie2-Cre and littermate controls (n = valves [mice], as indicated). Leaflet length = 0 corresponds to valves with no leaflets.

I Images of P7 intestine (on the left) and mesentery (on the right) showing submucosal reflux (arrows) and leakage (arrowheads) in the Foxp2flox/-;Tie2-Cre mice.

(J) Chyle phenotypes in P7 Foxp2flox/-;Tie2-Cre and littermate controls (n = number of mice as indicated).

Data information: In (E, G, H), data are presented as mean ± SD. P, Student's t-test. In (J), P, Fisher's exact test. Scale bar: 100 µm (B, D), 50 µm (C, F), 500 µm (I).
Transcription factor FOXP2 is a flow-induced regulator of collecting lymphatic vessels (2021)
Figure 4

Figure 4 - Downregulation of FOXP2 targets Nrp2 and Efnb2 in Foxp2 deficient lymphatic vessels.

A, B Whole-mount immunofluorescence (A) and quantification of NRP2 staining intensity (B) in P6 mesenteric lymphatic vessels of Foxp2flox/-;Tie2-Cre and littermate control mice (n = vessels [mice], as indicated).

C, D RNAscope-based whole-mount in situ hybridization (C) and quantification of Efnb2 transcript levels (D) in P7 mesenteric lymphatic vessels of Foxp2flox/-;Tie2-Cre and littermate control mice (n = vessels [mice], as indicated).

Data information: In (B, D), data are presented as mean ± SD. P, Student's t-test. Scale bar: 100 µm.
Transcription factor FOXP2 is a flow-induced regulator of collecting lymphatic vessels (2021)
Figure 5

Figure 5 - LEC-autonomous role of FOXP2 in lymphatic valve morphogenesis.

A Genetic constructs and 4-OHT treatments for neonatal LEC-specific Foxp2 deletion.

B Whole-mount immunofluorescence of P6 mesenteries, showing valve defects in the mutant vessels.

C-E Quantification of lymphatic valve numbers (C), valve leaflet length (D) and vessel width (E) in Foxp2flox/flox;Prox1-CreERT2 and littermate control mice (n = vessels (C, E) or valves (D) [mice], as indicated).

Data information: In (C-E), data are presented as mean ± SD. P, Student's t-test. ns = not significant. Scale bar: 200 µm.
Transcription factor FOXP2 is a flow-induced regulator of collecting lymphatic vessels (2021)
Figure 6

Figure 6 - FOXP2 is a component of the FOXC2/NFATc1 pathway.

A qRT-PCR analysis of FOXP2, FOXC2 and GJA4 expression in control (siCTRL) and FOXP2 or FOXC2 siRNA-treated HDLECs (n = 6 (FOXP2, FOXC2) or n = 3 (GJA4) independent experiments).

B Top panels: whole-mount immunofluorescence of E18 Foxc2flox/flox;Prox1-CreERT2 and littermate control mesenteries, showing downregulation of FOXP2 in Foxc2 deficient vessels. Bottom panels: extraction of nuclear FOXP2 staining (grey) using IMARIS surface mask generated based on PROX1 staining (green). Unmasked FOXP2 staining is shown in magenta.

C Quantification of FOXP2 levels in mesenteric lymphatic vessels of Foxc2flox/flox;Prox1-CreERT2 and control littermates based on nuclear FOXP2 signals as in (B) (n = vessels [mice], as indicated).

D Whole-mount immunofluorescence of P8 mesenteric lymphatic vessels of Foxp2flox/-;Tie2-Cre and littermate control mice showing loss of organized FOXC2high valve regions but unaltered expression of FOXC2 in Foxp2 deficient vessels.

E Whole-mount immunofluorescence of lymphatic valves in P7 mesenteries of control and Foxp2flox/-;Tie2-Cre mice showing NFATc1 subcellular localization. Note predominantly nuclear localization of NFATc1 in control valves (left panels), but cytoplasmic localization (middle panels, 3 out of 5 mice) or downregulation of NFATc1 (right panels, 2 out of 5 mice) in the mutant valves.

F Quantification of protein localization in NFATc1+ lymphatic valves from (D) (n = valves [mice], as indicated).

G Schematic of the molecular pathway involving FOXC2/FOXP2/NFATc1 in collecting vessel and valve morphogenesis.

Data information: In (A, C, F), data are presented as mean ± SD. P, One-way ANOVA (A), Student's t-test (C), Fisher's exact test (F). Scale bar: 50 µm (B, D, E).

Expanded View Figure legends
A whole genome scan for Artemisinin cytotoxicity reveals a novel therapy for human brain tumors (2022)
Figure 1

Figure 1. Mitochondria are key modulators of dihydroartemisinin toxicity in yeast.

A Schematic of yeast HIP-HOP and haploid embryonic stem cell screening for compound target identification.

B Haploinsufficiency profiling (HIP) of a yeast deletion collection at sub-lethal concentrations (IC30, 3μM) of dihydroartemisinin (DHA). Profile is plotted by sensitivity and z-score (i.e. relevance, for more information see materials and methods). Strains with essential genes are indicated in grey, relevant mutants of non-essential genes (black) are labelled.

C Homozygous profile (HOP) of the yeast deletion collection with DHA (IC10, 10μM). Sensitivity and z-scores are plotted.

D GO-term analysis of HIP-HOP hits.

E Mitochondrial (MitoTracker) staining of DMSO (upper panel) or DHA (lower panel) treated cells. Scale bar 5μm.

F Minimal inhibitory concentrations of DHA treated wild-type, heterozygous (YME1/yme1) and homozygous (yme1/yme1) mutant strains. Plates were imaged after 72h.

G HOP profile alignment of z-scores (significances) of DHA and acifluorfen. Z-scores couple the sensitivity score of a strain in a given profile to the variability in sensitivity of that strain across all the compounds tested.
A whole genome scan for Artemisinin cytotoxicity reveals a novel therapy for human brain tumors (2022)
Figure 3

Figure 3. Modulation of porphyrin biosynthesis is sufficient to alter dihydroartemisinin toxicity via ROS generation.

A, B Cell survival of dihydroartemisinin treated ESCs in combination with (A) the Ppox inhibitor acifluorfen or upon (B) 5-ALA administration. Alamar Blue staining was used to assess viability after 72h of treatment.

C, D Cell survival of DHA treated primary human glioblastoma cells from cancer patients and in combination with (C) the Ppox inhibitor acifluorfen (BTL90 cells) or upon (D) 5-ALA administration (VBT12 cells). Viability was assessed using CellTiter-Glo after 72h. Experiments were performed in triplicate. Values are mean ± SD.

E, F (E) ROS levels (DHE staining, PE 582/15 nm - MFI) and (F) cell survival of DHA (0.5μM), 5-ALA (0.25mM) or Ppox inhibitor (10μM) treated human neuroblastoma cells (SHSY5Y). Fluorescence of DHE (ROS levels) and cell numbers (survival) were assessed by flow cytometry and automated cell counting. The experiments were performed two times, in triplicate each. Values indicate mean ± SD. n.s. non-significant, **p < 0.01, ***p < 0.001, unpaired Student's t-test.
A whole genome scan for Artemisinin cytotoxicity reveals a novel therapy for human brain tumors (2022)
Figure 4

Figure 4. Cerebral tumor organoids (CNS-PNET-like) display increased sensitivity to dihydroartemisinin and 5-ALA combination therapy.

A Human cerebral tumor organoids for compound profiling.

B Schematic workflow of 5-ALA and DHA treated organoids.

C, D (C) Fluorescence and (D) brightfield images of DMSO (control), 5-ALA (0.0625mM), DHA (1μM), or 5-ALA and DHA (0.0625mM and 1μM) treated tumor organoids (CNS-PNET-like, c-MYC overexpression). Organoids were imaged on day 1, day 5 and day 7. One experiment is shown, representative of four independent experiments. Scale bar 500μm.

E Image analysis and quantification of GFP-positive tumor areas of the treated organoids. At least eight organoids per condition were analysed on day 3 and normalized to day 1. Three independent experiments were performed, with two to four organoids per batch, per condition. Data is shown as box plots (25th-75th percentiles, median).

F Flow cytometry analysis of dissociated cerebral tumor organoids. Relative percentages of GFP+ tumor cells of, normalized to control (DMSO), are shown on day 5. At least eight organoids per condition were analysed. Three independent experiments were performed using all conditions, with two to four organoids each per batch per condition. Box plots (25th-75th percentiles, median) of data are indicated.

G ROS/ DHE staining and flow cytometry analysis of dissociated tumor organoids (PE, 582/15 nm). Mean fluorescence intensities (MFI) of DHE stained GFP+ tumor cells and GFP- control cells are indicated. n=2, values are mean ± SD.

H Quantification of γH2AX+ cells in cerebral tumor organoids. 6 cryo-sections of 3 organoids each were stained for γH2AX, scanned and 25 regions of interest (ROI 2.500μm2) were analyzed per condition. Raw data were normalized to organoid area (per ROI) and are shown as box plots (25th-75th percentiles, median).

Data information: n.s. non-significant, *p < 0.05, unpaired Student's t-test.
Blocking mitochondrial pyruvate import in brown adipocytes induces energy wasting via lipid cycling (2020)
Figure 2

Figure 2: MPC1 inhibition stimulates mitochondrial respiration coupled to ATP synthesis in brown adipocytes.

(A,B) Effect of UK5099 treatment on norepinephrine-stimulated energy expenditure. Fully differentiated primary brown adipocytes were pre-treated with vehicle (DMSO) or 100 nM UK5099 for 2 h. OCR were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine in the presence of vehicle or UK5099. Norepinephrine (NE; 1 µM), oligomycin A (Oligo; 4 µM), etomoxir (Eto; 40 µM) and antimycin A (AA; 4 µM) were injected where indicated. (A) Representative OCR traces averaging 4 technical replicates. (B) Quantification of non-stimulated OCR, NE-stimulated OCR, ATP-linked OCR, mitochondrial proton leak and Eto-sensitive OCR as shown in (A) from n=9 individual experiments. The effects of UK5099 treatment were normalized to vehicle for each experiment. ** p < 0.01, *** p < 0.001, **** p < 0.0001 compared to vehicle by Student´s t test.

(C-H) Primary brown adipocytes were transfected with MPC1 siRNA (MPC1 KD) or Scramble RNA (Scramble). (C) Representative Western Blot analysis of MPC1, MPC2 and actin. (D) Quantification of MPC1 and MPC2 expression normalized to actin from Western Blots in (C) from n=3 individual experiments. ** p < 0.01 compared to Scramble by Students t test. (E) mRNA levels of MPC1 and MPC2 from n=3 individual experiments. **** p < 0.0001 compared to Scramble by Student´s t-test. (F) Representative OCR traces of differentiated primary brown adipocytes averaging 6 technical replicates. OCR were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine. Norepinephrine (NE; 1 µM), oligomycin A (Oligo; 4 µM), etomoxir (Eto; 40 µM) and antimycin A (AA; 4 µM) were injected where indicated. (G) Quantification of basal, NE-stimulated and Eto-sensitive OCR as shown in (F) from n=7 individual experiments. Data were normalized to Scramble RNA for each individual experiment. ns p>0.05, * p < 0.05, ** p < 0.01 compared to Scramble by Student´s t test. (H) Quantification of basal OCR in response to 100 nM UK5099 treatment in Scramble RNA of MPC1 siRNA transfected cells from n=5 individual experiments. Data were normalized to vehicle for each experiment. Note that UK5099 capacity to increase OCR in non-stimulated brown adipocytes is absent in cells where MPC1 is downregulated. ns p>0.05, * p < 0.05 compared to vehicle by Student´s t test.

Data information: All data are presented as mean ± SEM.
Blocking mitochondrial pyruvate import in brown adipocytes induces energy wasting via lipid cycling (2020)
Figure 3

Figure 3: Glutamine metabolism is required for increased energy expenditure induced by MPC inhibition

(A,B) Requirement for glutamine in UK5099-induced energy expenditure. Primary brown adipocytes were pre-treated with vehicle (DMSO) or 100 nM UK5099 for 2 h. OCR were measured in presence of either 5 mM glucose and 3 mM glutamine or 5 mM glucose alone. Norepinephrine (NE; 1 µM), oligomycin A (Oligo; 4 µM), etomoxir (Eto; 40 µM) and antimycin A (AA; 4 µM) were injected where indicated. (A) Representative OCR traces averaging 6 technical replicates. (B) Quantification of non-stimulated OCR, NE-stimulated OCR, ATP-linked OCR, mitochondrial proton leak and etomoxir-sensitive OCR after vehicle or UK5099 treatment as shown in (A) from n=4 individual experiments. Data were normalized to vehicle for each experiment. Note that UK5099 treatment increased OCR only when cells were assayed in the presence of both glucose and glutamine. * p < 0.05, compared to vehicle by Student´s t test.

(C) Effect of glutaminase inhibitor on UK5099-induced energy expenditure. Primary brown adipocytes were pre-treated with either vehicle (DMSO), 100 nM UK5099, or 100 nM UK5099 + 2.5 µM CB839 for 2 h. OCR were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine in the presence of vehicle, UK5099 or UK5099 + CB839 from n=5 individual experiments. Data were normalized to vehicle for each experiment. * p < 0.05, ** p < 0.01 by ANOVA.

(D) Effect of MPC inhibition on glutamate/aspartate ratio measurement. Glutamate/aspartate ratio was quantified using GC-MS as a measure of glutamine catabolism. Primary brown adipocytes were treated for 24 h with vehicle (DMSO) or 10 µM UK5099 from n=6 individual experiments. ** p < 0.01, compared to vehicle by Student´s t test.

Data information: All data are presented as mean ± SEM.
Unbiased proteomic profiling of host cell extracellular vesicle composition and dynamics upon HIV-1 infection (2021)
Figure 3

Figure 3. Biochemical analysis of the composition of EVs released by Jurkat cells.

EVs were purified by SEC from supernatants of Jurkat cells.

(A) EVs were subjected to immuno-isolation with beads coupled to antibodies against CD81 or CD63. Bead-associated (Pull-down: PD) vesicles and those left behind (Flow-Through: FT) were loaded on a gel for Western blot analysis with antibodies specific for CD63, Syntenin-1, CD81, ADAM10 and CD3G. A representative blot (top panel) and quantification (mean ± SD) of the proportion of signal in FT as compared with total (PD+FT) from four independent experiments (bottom panel) are shown.

(B) Representative images of double-immunogold labelling of EVs purified by SEC from Jurkat cells. Left panel, CD81/CD63; right panel, CD81/CD3E. Arrows show CD63 staining in CD81+CD63+ EVs.

(C,D) Multiplex bead-based flow cytometry assay for detection of EV surface markers. Antibody-coated capture beads were incubated with 2 x 109 particles. Captured EVs were detected with either APC-labeled anti-CD81, anti-CD63 or anti-CD3E. (C) Median APC fluorescence values for the different bead populations is shown as the ratio to the median APC fluorescence of control beads (log10 scale). Mean ± SD for four independent experiments is shown. (D) Heat-map representation of the median APC fluorescence values for the different bead populations detected with anti-CD63 or anti-CD3E antibodies relative to the values detected with anti-CD81. Mean of four independent experiments.