EMBO/soda-nel huggingface dataset:
MARCO+ lymphatic endothelial cells sequester arthritogenic alphaviruses to limit viremia and viral dissemination (2021)
Figure 7

Figure 7. MARCO+ LECs harbor CHIKV RNA.

WT C57BL/6 mice were s.c. inoculated with PBS (mock, n=3) or 103 PFU of CHIKV (n = 3) in the left rear footpad. At 24 hpi, the dLN was collected and enzymatically digested into a single cell suspension. Cells were enriched for CD45- cells and analyzed by scRNA-seq as described in the materials and methods.

(A) UMAP projection shows each replicate for mock- and CHIKV-infected mice; the number of cells obtained for each replicate is shown at the bottom.

(B) UMAP projection shows annotated cell types (top) and the proportion of cells identified for each cell type (bottom).

(C) UMAP projection shows endothelial subtypes (top) and the proportion of cells identified for each cell type (bottom). Non-endothelial cells are shown in white.

(D) UMAP projection shows Marco expression.

(E) UMAP projection shows the fraction of counts that align to the CHIKV genome.

(F) UMAP projection shows cell types for cells classified as CHIKV-high. CHIKV-low cells are shown in white. The proportion of CHIKV-high cells belonging to each cell type is shown on the right.

(G) The fraction of counts that align to the CHIKV genome is shown for CHIKV-high cells. Only cell types that include >20 cells are shown.

(H, I) MARCO (H) and Mxra8 (I) expression is shown for MARCO+ LECs for mock-infected cells and CHIKV-infected cells classified as either CHIKV-low or CHIKV-high. P values were calculated using a two-sided Wilcoxon rank-sum test with Bonferroni correction. In the boxplot, the central lines, the box limits, and the whiskers represent medians, the interquartile range (IQR), and min/max values that are not outliers, respectively. Outliers are shown as points and include any values that are more than 1.5x IQR away from the box.

Expanded View Figure Legends
Autophagy in thymic epithelium shapes the T-cell repertoire and is essential for tolerance (2008)
figf1

a, Sections of the thymus and muscle of fed and starved GFP–LC3 mice were stained with anti-GFP Alexa 488 antibody (green) and 4,6-diamidino-2-phenylindole (DAPI; blue). Images are representative of n = 6. b, Thymic stromal cells from GFP–LC3 mice were fixed onto glass slides and stained with anti-GFP Alexa 488 and DAPI. Typical autophagy-positive cTECs and mTEChi are shown. The average frequencies ± s.d. of cTECs and mTEChi scoring autophagy positive (that is, ≥5 autophagosomes) in two independent preparations are indicated. Less than 1% of dendritic cells (DCs) and mTEClo were positive. c, Relative distribution of the number of autophagosomes in autophagy-positive cTECs and mTEChi; a.u., arbitrary units.
Autophagy in thymic epithelium shapes the T-cell repertoire and is essential for tolerance (2008)
figf2

a, Thymi from wild-type (WT) or Atg5–/– embryos were transplanted under the kidney capsule of wild-type recipients and analysed 6 weeks later. The average cellularities ± s.d. are shown; n = 5. b, Normal compartmentalization of Atg5–/– grafts in cytokeratin-8-positive (red; K8) cortical and cytokeratin-5-positive (green; K5) medullary regions; n = 8. c, Expression of MHC class I and II on cTECs or mTECs from Atg5–/– to wild-type (WT) grafts (red), or wild-type to wild-type grafts (blue). Staining controls are depicted in grey; data are representative of three experiments. d, RT–PCR analysis of autoimmune regulator (Aire) and TRA expression in mTECs isolated from Atg5–/– to wild-type, or wild-type to wild-type grafts. Expression of &bgr;-actin (Actb), intestinal trefoil factor (Tff3), casein 2 (Csn2) and salivary protein 1 (Spt1) are also shown (5-fold serial dilutions). Data are representative of two experiments.
Autophagy in thymic epithelium shapes the T-cell repertoire and is essential for tolerance (2008)
figf3

a, Polyclonal T-cell development in Atg5–/– to wild-type (WT), or wild-type to wild-type grafts. The top dot plots show CD4 and CD8 staining; the bottom dot plots show expression of CD25 and Foxp3 by CD4 SP thymocytes; n ≥ 5. b, Atg5–/– or wild-type lobes were grafted into TCR-HA transgenic recipients. Graft cellularity ± s.d. and the expression of the transgenic TCR on CD4 SP cells were analysed after 6 weeks. The frequency ± s.d. of TCR-HA+ cells among CD4 SP cells was 32.1% ± 3.2% in wild-type to TCR-HA, and 19.6% ± 4.3% in Atg5–/– to TCR-HA lobes; P = 1.8 × 10-8 (n ≥14). c, Atg5–/– or wild-type lobes were grafted into AND transgenic mice. Graft cellularity ± s.d. and the expression of the transgenic TCR (V&bgr;3–V&agr;11) on CD4 SP cells were analysed after 6 weeks. The frequency ± s.d. of V&bgr;3+ V&agr;11+ CD4 SP cells was 93.5% ± 0.2% in wild-type to AND lobes (n = 3) and 97.9% ± 1.1% (n = 6) in Atg5–/– to AND lobes (P = 9 × 10-4). d, Thymi from wild-type or Atg5–/– embryos were grafted into P14, OT-I or HY TCR-transgenic recipients. The dot plots show the expression of the transgenic TCR V-segments on CD8 SP cells. Thymus cellularities ± s.d. are shown in the respective bar diagrams. Data are representative of n = 4 (P14), n ≥ 4 (OT-I) and n ≥ 2 (HY). e, Thymi from wild-type or (BALB/c × C57BL/6)F1 Atg5–/– embryos were transplanted into (BALB/c × C57BL/6)F1 recipients. Six weeks after grafting, expression of the I-E&agr;52–68–I-Ab complex on cTECs was analysed using the monoclonal antibody Y-Ae. Atg5–/– cTECs are shown in red; wild-type cTECs are shown in blue. Control stainings are shown in orange (Atg5–/–) and green (wild type). Data are representative of two independent experiments.
Autophagy in thymic epithelium shapes the T-cell repertoire and is essential for tolerance (2008)
figf4

Atg5–/– or wild-type (WT) lobes were grafted into athymic (nu/nu) mice (nu/nuAtg5–/– or nu/nuWT, respectively). Data are representative of three experiments with n ≥ 4. a, Expression of activation markers on lymph node CD4 T cells in nu/nuAtg5–/– or nu/nuWT chimaeras. b, The changes in body weight over time are shown. c, The appearance of nu/nuAtg5–/– or nu/nuWT chimaeras. The colon (arrow) and uterus (arrowhead) are highlighted; note the absence of fat pads (asterisk) in nu/nuAtg5–/– chimaeras. d, Haematoxylin and eosin staining of organs from nu/nuAtg5–/– or nu/nuWT chimaeras. Arrows indicate infiltrates.
Molecular pathways of senescence regulate placental structure and function (2019)
Figure 1

Figure 1. Senescence Pathways are Downregulated in Human Placentas Complicated by IUGR

(A-B) Human placental syncytiotrophoblast cells express markers of cellular senescence, p16 and p21. Sections of normal human post-partum, third trimester, placenta were evaluated by immunofluorescence co-staining for senescence regulators (A) p16 and (B) p21. Green label: the syncytiotrophoblast marker βHCG, red label: p16 or p21. Blue label: DAPI nuclear stain. Arrowheads indicate positively stained syncytiotrophoblast cells βHCG+/p16+ (A), βHCG+/p21+ (B). Scale bar, 50 µm.

(C-F) Sections of human normal third-trimester placentas post-partum (n=4 normal placentas) and IUGR-complicated placentas of the same gestational age (n=4 IUGR placentas) were stained for senescence-related markers of p15 (C), p16 (D), p21 (E), and p53 (F) in the syncytiotrophoblast (n=3 sections, derived from each normal and IUGR placenta). Percentages of positively stained syncytia were quantified (n= at least 12 fields of view from all sections). Scale bar, 100 µm. Arrowheads indicate syncytiotrophoblast cells.

(G) Immunoblot analysis of the protein content of p16, p21, p53 and DCR in normal (n=4 placentas) and in IUGR-complicated placentas (n=4 placentas). Each lane represents one independent placenta. (H) Quantitative RT-PCR analysis of p16, p21 and DCR2 expression in IUGR-complicated and in normal placentas. Results were obtained from four human normal and four IUGR placentas.

Data information: Values are means + SEM of at least three experimental repeats. Statistical significance was determined by unpaired two-tailed Student's t-test (*) p < 0.05; (**) p < 0.01; (***) p < 0.001.
Molecular pathways of senescence regulate placental structure and function (2019)
Figure 2

Figure 2. Placentas with Attenuated Senescence Programs Exhibit Altered Signal Intensity Dynamics on In-Utero DCE-MRI

DCE-MRI was performed on pregnant mice of WT, Cdkn1a, p53, Cdkn2a and Cdkn2a;p53 genotypes on day E14.5. Mice were injected i.v. with an albumin-labeled contrast agent (biotin-BSA-GdDTPA; 10 mg/mouse) and placental enhancement was monitored at 9.4T MRI for 60 min.

(A) Representative T1-weighted image of a pregnant WT mouse and a selection of four different placentas (outlined by colored lines).

(B) Representative SI dynamics of a single WT placenta (marked in A), following biotin-BSA-GdDTPA administered for 60 min. SI parameters of Initial Enhancement and Recovery are illustrated.

(C) Representative SI dynamics of a single WT placenta (marked in A) over time. Scale bar, 2.5 mm.

(D−G) Representative SI plots of Cdkn1a-/- (D), p53-/- (E), Cdkn2a-/- (F), and Cdkn2a-/-;p53-/- (G) placentas, compared to their WT or HET littermates.

(H−I) Quantification of the SI Initial Enhancement (H) and SI Recovery (I) in WT, Cdkn1a-/-, p53-/-, Cdkn2a-/-, and Cdkn2a-/-;p53-/- placentas. MRI experiments were repeated at least three times for each murine genotype (n>=3 placental SI measurements from each genotype). Values are means +SEM; Statistical significance was determined by unpaired two-tailed Student's t-test (*) p < 0.05; (**) p < 0.01; (***) p < 0.001.
Molecular pathways of senescence regulate placental structure and function (2019)
Figure 3

Figure 3. Murine Placentas with Attenuated Senescence Programs Exhibit Morphological Anomalies in the Labyrinth

Histological evaluation of H&E-stained sections in the labyrinth zone of murine Cdkn2a-/- (n=5 placentas), Cdkn2a-/-;p53-/- (n=5), p53-/- (n=4), and Cdkn1a-/- (n=3) placentas on day E14.5, compared to WT placentas (n=6).

(A−E) The labyrinth zone of the WT (A) differs from those of Cdkn2a-/- (B) and Cdkn2a-/-;p53-/- (C) placentas. Black arrows indicate regular-sized nuclei in the WT placenta and polyploidy in the Cdkn2a-/- and Cdkn2a-/-;p53-/- placentas. Blue arrows indicate normal vasculature in the WT placenta and collapsed vasculature in the Cdkn2a-/- and Cdkn2a-/-;p53-/- placentas. Labyrinths of p53-/- (D) and Cdkn1a-/- (E) placentas did not differ from that of the WT placenta. L, labyrinth. Staining for each placenta was performed in triplicates.

(F−G) Ki67 immunostaining in the labyrinth zones of murine WT , p53-/- ,Cdkn2a-/-, and Cdkn2a-/-;p53-/- placentas, on day E14.5 (n=3 placentas from each genotype). (F) Representative images of Ki67 staining in the labyrinth zone are shown. Positive Ki67 trophoblast cells are indicated with arrows. L, labyrinth. (G) Proliferation scores (n=3 scores, derived from three placentas of each genotype) based on Ki67 staining show enhanced Ki67 expression in the placental labyrinths of Cdkn2a-/- and Cdkn2a-/-;p53-/- placentas relative to WT. Values are means +SEM of three scores for each genotype. *P < 0.05 by a one-tailed unpaired Student's t-test.

(H) Representative images of SA-β-gal staining in the labyrinth zones of WT, Cdkn2a-/-, and Cdkn2a-/-;p53-/- placentas on day E14.5.

(I) SA-β-gal activity, quantified by ImageJ sofware in Cdkn2a-/- and Cdkn2a-/-;p53-/- labyrinths, is significantly reduced relative to those in WT labyrinth (n>=7 fields of view from each genotype). Values are means + SEM. (***) p < 0.001 by a two-tailed unpaired Student's t-test.

Data information: Scale bars, 50 µm.
Molecular pathways of senescence regulate placental structure and function (2019)
Figure 4

Figure 4. Human Primary Syncytiotrophoblasts Activate Regulatory Pathways of Senescence

Human term placentas were dissected and cytotrophoblast cells were extracted and seeded.

(A, B) On days 1 and 5 after seeding, cell fusion was monitored by staining for phalloidin (F-actin) (green) and DAPI (blue). Scale bar, 50 μm. (B) Trophoblast cell fusion was quantified on days 1, 3, and 5 post-seeding. Values are means + SEM, derived independently from human placentas of three pregnancies.

(C) Quantification of the human βhCG hormone, normally secreted by functional syncytiotrophoblast cells, in the medium of a representative trophoblast culture on post-delivery days 2, 3 and 5.

(D) Gene set enrichment analysis (GSEA) of published datasets from human primary trophoblast cultures on days 5 and 2 (n=4 trophoblast culture of day 5 and day 2). Gene sets related to pregnancy, aging, immune response, cytokine activity, MAPK, JAK-STAT, NF-κB and metalloproteinase activity were found to be enriched in day 5 cultures, whereas on the same day gene sets related to cell cycle, E2F targets, mitosis and DNA replication were downregulated. Statistical significance was determined by permutation testing with normalized enrichment score (NES)

(E, F) Quantitative RT-PCR analysis of expression of the cell cycle-related genes p16, p21 and CCNE1 (E) and of the syncytiotrophoblast markers GCM1, CGA and CGB (F).

(G) Immunoblot analysis of the indicated proteins in human trophoblast cells, derived independently from two placentas, on days 2 and 5.

(H, I) Quantitative RT-PCR analysis of expression of the SASP components CCL2, CCL5, CCL8, IL6, IL1β, TGF-β and PDG-FC (H), and of the metalloproteases MMP2, MMP3 and MMP9 (I), in human primary trophoblast cultures on days 5 and 2.

Data information: Quantitative RT-PCR values are expressed as means +SEM. Results were obtained from trophoblast cultures derived from human placentas of four independent pregnancies. All RT-PCR experiments were performed in triplicates and repeated at least three times. Statistical significance was determined by unpaired two-tailed Student's t-test (*) p < 0.05; (**) p < 0.01.
Molecular pathways of senescence regulate placental structure and function (2019)
Figure 5

Figure 5. Senescence Pathways Regulate Gelatinase Expression

(A) Immunoblot analysis of the indicated proteins in WT and Cdkn2a-/-;p53-/- placentas, on day E14.5 (two independent placentas out of four from each genotype are shown).

(B, C) RT-PCR analysis of expression of Mmp2 and Mmp9 in murine Cdkn2a-/-;p53-/- and in WT placentas on day E14.5 (n>=4 fields of view for each genotype). Values are means + SEM of at least three experimental repeats.

(D, E) Representative images (D) and quantification (E) of in-situ gelatin zymography of the labyrinth zone of murine WT and Cdkn2a-/-;p53-/- placentas on day E14.5. Scale bar, 50 μm.

(F, G) Representative images (F) and quantification (G) of in-situ zymography of human normal third trimester post-partum placentas and placentas from IUGR-complicated pregnancies of the same gestational age (n>=9 fields of view of normal and IUGR placentas). Scale bar, 100 µm.

(H) Immunoblot analysis of the protein content of p-p65, p65, p-STAT3 and STAT3 in normal placentas (n=4) and placentas from IUGR-complicated pregnancies (n=4). Each lane represents one independent placenta.

(I) Quantitative RT-PCR analysis of expression of MMP2, MMP9, IL6, and the JAK-STAT targets IFNAR1 and IFNAR2, in normal placentas and placentas from IUGR-complicated pregnancies. Results are from four human normal placentas and four placentas from IUGR-complicated pregnancies.

Data information: Values are means + SEM of at least three experimental repeats. Statistical significance was determined by unpaired two-tailed Student's t-test (*) p < 0.05; (**) p < 0.01; (***) P<0.001.
Sex differences and risk factors for bleeding in Alagille syndrome (2022)
Figure 2

Figure 2. Cholestatic Jag1Ndr/Ndr mice exhibit normal coagulation but a thinner skull and sporadic spontaneous and provoked hemorrhages.

A Pup survival analysis between P0-P10, by sex. Each dot represents the absolute percent of remaining animals per group (Jag1CTRL n=211, male Jag1Ndr/Ndr n=26, female Jag1Ndr/Ndr n= 20, log-rank test, ****P<0.0001).

B P10 plasma total bilirubin levels (n=6 per group, unpaired t test, **P=0.0091).

C P10 plasma thrombin antithrombin levels (n=6 per group, unpaired t test, ns, P=0.2788).

D P10 serum fibrinogen levels (n=6 per group, unpaired t test, ns, P=0.7585).

E P10 whole blood prothrombin time (INR) (n=6 per group, unpaired t test, ns, P>0.9999).

F P10 tail bleeding time (n=6 per group, unpaired t test, ns, P=0.8916).

G-I µCT of P30 skulls. (G) Color map depicting wall thickness. Scale bar 3.5 mm. (H) Skull full thickness volume (n=5-6 per group, two-way ANOVA. Interaction P= 0.7595; Sex P= 0.1164, Genotype **P= 0.0012. Šídák's multiple comparisons test: Male Jag1+/+ vs Jag1Ndr/Ndr *P= 0.0227; : Female Jag1+/+ vs Jag1Ndr/Ndr *P= 0.034), (I) Compact bone total volume (n=5-6 per group, two-way ANOVA. Interaction P= 0.8631; Sex P= 0.0901, Genotype **P= 0.0014. Šídák's multiple comparisons test: Male Jag1+/+ vs Jag1Ndr/Ndr *P= 0.0305; Female Jag1+/+ vs Jag1Ndr/Ndr *P= 0.0307).

J-K (J) Provoked vascular accidents induced by portal vein resin injection, with (K) resin leakage quantification outside of blood vessels (red) in adult mice (n=6 per group, unpaired t test, ns, P=0.1088). Scale bar 1 mm.

L Adult brain Evans blue assay (Jag1CTRL n=7, Jag1Ndr/Ndr n=8, unpaired t test, ns, P=0.3807).

M Hemorrhagic P3 Jag1Ndr/Ndr brain (n= 2/83 Jag1Ndr/Ndr mice, sex n.d.).

N Hemorrhagic P15 Jag1Ndr/Ndr retina. Scale bar 20 µm. Sex n.d.

O Red blood cells outside P10 Jag1Ndr/Ndr retinal arteriole. Scale bar 10 µm.

Data information: Bar graphs depict mean values ± standard deviation, each dot represents one biological replicate. Circles represent females, squares represent males. For details/results of statistical analyses, please see source data. µCT, micro computed tomography; n.d., not determined; OD, optical density; TAT, thrombin antithrombin; P(X), postnatal day X.
Sex differences and risk factors for bleeding in Alagille syndrome (2022)
Figure 5

Figure 5. Capillary homeostasis is compromised, and retinal ganglion cells break down in Jag1Ndr/Ndr retinas.

A-D (A) P30 retina, three CD31+ capillary layers pseudo-colored for superficial, intermediate and deep layers. Scale bar 50 µm. (B) P30 SCP branching point number (n=6-8 per group, unpaired t test, ns, P=0.5346). (C) P30 ICP vascular length (n=6 per group, unpaired t test, *P=0.0124). (D) P30 ICP branching point number (n=6 per group, unpaired t test, **P=0.0057).

E-H (E) 3-6 months old retina, three pseudo-colored CD31+ capillary layers. Scale bar 50 µm. (F) Adult SCP branching point number (n=6 per group, unpaired t test, ns, P=0.5365). (G) Adult ICP vascular length (n=6-7 per group, unpaired t test, **P=0.0078). (H) Adult ICP branching point number (n=6-7 per group, unpaired t test, **P=0.001).

I-J (I) ICP COLIV and CD31 (arrowhead labels COLIV+ CD31- capillary). Scale bar 50 µm. (J) Empty COLIV sleeve length in ICP per field (n=6 per group, unpaired t test, ns, P=0.1583).

K-M (K) Retinal capillary plexuses in 3-6 months old (top panels) and one-year-old mice (bottom panels). White arrowheads label discontinuous ICP. Blue arrowhead labels SCP, green ICP and magenta DCP. (L-M) Vertical sprout number between SCP (S) and ICP (I) and ICP (I) and DCP (D) in (L) adult (n=6 per group, two-way ANOVA, genotype ***P=0.0004, Sidak's multiple comparisons test S-I ns, P=0.0592, I-D ****P<0.0001) and (M) one-year-old mice (n=8 per group, two-way ANOVA, genotype ns, P=0.4507, Sidak's multiple comparisons test S-I ns, P=0.9483, I-D ns, P=0.6046).

N-P (N) P10 and (O) P40 neurofilament (NF) and CD31 staining. Scale bar 50 µm. (P) % area positive for NF in P10 and P40 mice (n=6 per group, two-way ANOVA: Interaction ***P= 0.0001; Age *P=0.0428; Genotype ***P=0.0004. Sidak's multiple comparisons test: P10 Jag1+/+ vs P10 Jag1Ndr/Ndr P= 0.9246; P40 Jag1+/+ vs P40 Jag1Ndr/Ndr ****P= <0.0001).

Data information: COLIV, collagen IV; D or DCP, deep capillary plexus; I or ICP, intermediate capillary plexus; NF, neurofilament; S or SCP, superficial capillary plexus; P(X), postnatal day X.

Bar graphs depict mean values ± standard deviation, each dot represents one biological replicate. Circles represent females, squares represent males. For details/results of statistical analyses, please see source data.
PRC1-labeled microtubule bundles and kinetochore pairs show one-to-one association in metaphase (2016)
Figure 1

Figure 1. Most PRC1-labeled overlap bundles are coupled with sister kinetochores in metaphase.

A. Spindle in a fixed HeLa cell expressing PRC1-GFP (green) and mRFP-CENP-B (magenta) oriented horizontally with respect to the imaging plane, as shown in the scheme. Images of different z-slices (central plane of the spindle z=0, two images below, z=-4 µm and z=-2 µm, and above, z=+3 µm and z=+5 µm), maximum projection of a z-stack (max z), and 3D projections (3D) with the 3D coordinate system represented as a cuboidal box that indicates different spindle orientations are shown. Additional z-slices of this spindle are shown in Fig EV1A.

B. Spindle in a fixed HeLa cell expressing PRC1-GFP (green) and mRFP-CENP-B (magenta) oriented vertically with respect to the imaging plane, as shown in the scheme. Legend as in A. Additional z-slices of this spindle are shown in Fig EV1B.

C. Correlation between the number of PRC1-labeled bundles and the number of pairs of sister kinetochores counted throughout horizontal (black) and vertical (blue) spindles of fixed HeLa cells in metaphase. Data points represent individual spindles, lines show linear fits.

D. Pie charts showing the fraction of PRC1-labeled bundles associated with kinetochore pairs (blue), PRC1-labled bundles not associated with kinetochores (green) and kinetochores not associated with PRC1 bundles (magenta) in horizontal (top row) and vertical spindles (bottom row) from cells with up to 70 chromosomes (left column) and more than 70 chromosomes (right column). Horizontal spindles contained a total of 1786 kinetochore-PRC1 pairs (n=29 cells) and vertical spindles 1133 kinetochore-PRC1 pairs (n=16 cells).

E, F. Spindle length and spindle width as a function of the number of kinetochore pairs coupled with PRC1-labeled bundles.

G. Number of kinetochore pairs coupled with PRC1-labeled bundles per unit area of the cross section of the central part of horizontal (black) and vertical (blue) spindles as a function of the number of kinetochore pairs coupled with PRC1-labeled bundles in the spindle.

Scale bars, 2 µm; n, number of cells; R2, coefficient of determination; p, p-value from a t-test.