EMBO/soda-nel huggingface dataset:
Eosinophil-derived IL-4 is necessary to establish the inflammatory structure in innate inflammation (2022)
Figure 4

Figure 4: Eosinophil depletion delays resolution of zymosan-induced inflammation.

(A-C) FACS analysis of eosinophils in the blood (panel A,B; n= 6 mice) or paws (panel C n= 8 mice) at the indicated time points after zymosan injection. Anti-Siglec F antibody or the IgG-control (control) were administered with 0,883mg/kg 24 hours prior zymosan injection. Data are shown as mean ± SEM, student's t-test *p<0.05, **p<0.01, ***p<0.001 vs. control mice.

(D) Paw volume after injection of zymosan. Data are presented as mean ± SEM (n=8-10 mice). Two-way ANOVA/Bonferroni, ***p< 0.001.

(E, F) Zymosan-induced mechanical (panel E) and thermal (panel F) hypersensitivity of control or anti-Siglec F antibody-treated mice. Data are shown as the mean ± SEM (n=8 mice). Two-way ANOVA/Bonferroni, **p< 0.01, ***p< 0.001 vs. control mice.
Eosinophil-derived IL-4 is necessary to establish the inflammatory structure in innate inflammation (2022)
Figure 6

Figure 6: IL-4 mediates eosinophil effects during resolution of inflammation.

(A) IL-4 levels in paws from naive and zymosan-injected mice were determined by ELISA 8 and 24 hours after injection of zymosan in paws of control or eosinophil-depleted mice. Data are shown as mean ± SEM (n=6 mice). Student's t-test/Two-way ANOVA/Bonferroni, control vs anti-Siglec F antibody treatment: **p<0.01, ***p< 0.001.

(B) FACS analysis of the eosinophil number in paws from control and eosinophil-depleted mice with or without IL-4c treatment 24h prior zymosan injection. Data are shown as the mean ± SEM (n=8 mice). Two-way ANOVA/Bonferroni ***p< 0.001.

(C) Same as panel B except that resident (Ly6C-) and monocyte-derived macrophages (Ly6C+) were determined.

(D-F) Same as panel B except that M2-like (panel D), M1-like (panel E) and M0 macrophages (panel F) were determined. Data are shown as mean ± SEM (n=8 mice). Student's t-test/Two-way ANOVA/Bonferroni, control vs anti-Siglec F antibody treatment: *p<0.05, ***p<0.001.
Eosinophil-derived IL-4 is necessary to establish the inflammatory structure in innate inflammation (2022)
Figure 7

Figure 7: Eosinophil depletion increases neutrophil recruitment and lowers efferocytosis.

(A) Concentrations of cytokines and chemokines were determined by multiplex cytokine assay 8 and 24 hours after injection of zymosan in paws from control or eosinophil-depleted mice. Data are mean ± SEM (n=6 mice). Student's One-way ANOVA, significance between control and anti-Siglec F antibody treatment *p<0.05; **p<0.01; ***p<0.001.

(B,C) FACS analysis of the neutrophil count in paws (panel B) and peripheral blood (panel C) from control or eosinophil-depleted mice with or without IL-4c treatment. Data are shown as the mean ± SEM (n=6-8). Two-way ANOVA/Bonferroni, **p<0.01, ***p<0.001.

(D) Same as panel B except that efferocytosis was determined. *p<0.05; ***p<0.001

(E) FACS analysis of alive and dying neutrophils in paws from control or eosinophil-depleted mice. Data are shown as the mean ± SEM (n=5 mice).
Eosinophil-derived IL-4 is necessary to establish the inflammatory structure in innate inflammation (2022)
Figure 8

Figure 8: IL-4 mediates eosinophil effects on the inflammatory structure.

(A) Composite MELC image showing the disappearance of the pro- (PI) and the anti-inflammatory (AI) regions 24 hours after zymosan injection. Shown are representative images of mice receiving anti-Siglec F or IL-4c. The red dotted lines depict the area where the transition between the neighboring regions occurs.

(B) Relative distance of various immune cell types based on the likelihood for a direct neighborhood of macrophage subtypes in regard to zymosan at the 24 hours after zymosan injection. Data are shown as the mean ± SEM (n=5 mice).

(C) Zymosan-induced thermal hypersensitivity in control or eosinophil-depleted mice with or without IL-4c treatment 24h hours prior zymosan injection. The right panel show the comparison of paw withdrawal latencies between the 4 treatment groups at the indicated time points. Data are shown as the mean ± SEM (n=6 mice). Two-way ANOVA/Bonferroni, *p< 0.05, **p< 0.01; ns, not significant.

Figure Legends: Expanded version
ARGONAUTE 6 bridges transposable element mRNA‐derived siRNAs to the establishment of DNA methylation (2014)
Figure 1

A system to detect de novo TE DNA methylation guided by 21–22nt endo‐siRNAs

Bisulfite DNA methylation sequencing of the Athila6A TE transcriptional start site (TSS) in genotypes with silenced TEs (left of the dashed line) or transcriptionally active TEs (right of the dashed line). Methylation in the CG (red), CHG (blue) and CHH (green) sequence contexts are shown (H = C, T, or A). Error bars represent Wilson score interval 95% confidence limits. For multiple statistical tests, one‐way ANOVA was performed using Tukey's multiple comparison test. ns, not significant.
The reads‐per‐million‐(RPM)‐normalized accumulation of Athila6A TE siRNAs from TE‐silenced epigenomes (left) and ddm1 TE‐active epigenomes (right). Inset line graphs reflecting the same values are shown for select genotypes. All line graphs have the same y‐axis scale and are shown for increased resolution in genotypes with low siRNA levels.
ARGONAUTE 6 bridges transposable element mRNA‐derived siRNAs to the establishment of DNA methylation (2014)
Figure 2

AGO6 incorporates 21–22nt siRNAs

AGO6 relative enrichment of small RNAs (solid lines) and total small RNAs (dashed lines) are shown for each small RNA size class (21, 22 and 24nt) at four genomic loci.

A representative microRNA, miR158B. The location of miR158B from this locus is shown in orange.
The SimpleHat2 non‐autonomous TE targeted by Pol IV‐RdDM.
The RDR6‐RdDM target locus TAS3a.
The Athila6A consensus element in the wt Col epigenome.
The same Athila6A consensus element as in (D) upon transcriptional activation in the ddm1 epigenome.
The total small RNAs produced in the wt Col TE‐silenced epigenome.
The total small RNAs in ddm1 mutants.
Bisulfite sequencing of different regions of the Athila6A element. Error bars represent Wilson score interval 95% confidence limits. Differences in the methylation status were analyzed with a two‐tailed unpaired Student's t‐test.
A second biological replicate of the FLAG‐AGO6‐IP and no‐antigen control followed by deep sequencing shown for the Athila6A consensus element in ddm1 mutants. The transcriptional start sites of the LTR TSS‐ and IR‐driven transcripts are shown.
Analysis of the first base of AGO6‐enriched siRNAs from the wt Col TE‐silenced epigenome (left) and the ddm1 TE‐transcriptionally active epigenome (right).

Data information: Maps of the Athila6A element shown below panels (F) and (G) align with AGO6 enrichment shown in (D) and (E).
ARGONAUTE 6 bridges transposable element mRNA‐derived siRNAs to the establishment of DNA methylation (2014)
Figure 3

21–22nt siRNAs guide AGO6 to target chromatin

A Schematic of the ChIP crosslinking between chromatin and the AGO6 protein which is tethered to its targets by a Pol V‐derived RNA scaffold.
B H3Ac control ChIP from the same chromatin used in the AGO6‐IP for the At1g08200 constitutively expressed gene and the Athila6A TSS. Enrichment of H3Ac at the Athila6A TSS is increased upon transcriptional activation in ddm1 mutants, while enrichment of H3Ac at the gene At1g08200 is consistently high in both wt Col and ddm1. Data are represented as mean ± SD of six biological replicates for each genotype.
C–E AGO6‐ChIP qPCR analysis of (C) the gene At1g08200 and the Pol IV‐RdDM target SimpleHat2, (D) the Athila6A TSS in wt Col TE‐silenced epigenomes (left) and ddm1 TE‐active epigenomes (right) and (E) TAS3a. ChIP signals in (D) are normalized to the ago6 or ddm1 ago6 no‐antigen controls. ChIP signals in (C) and (E) are normalized to the ago6 no‐antigen control. In (C–E), each biological replicate data point is shown as a gray dot. The data are represented as mean ± SD. For multiple statistical tests, one‐way ANOVA was performed using Tukey's multiple comparison test.
F ChIP qPCR analysis of the FLAG‐tagged POL V protein in wt Col and ddm1. ChIP signals are normalized to the wt Col no‐antigen control.

Data are represented as mean ± SD of three biological replicates for each genotype.
ARGONAUTE 6 bridges transposable element mRNA‐derived siRNAs to the establishment of DNA methylation (2014)
Figure 4

Genome‐wide TE targets of RDR6‐RdDM

A AGO6 enrichment and depletion of 21–22nt siRNAs for the entire genome in 100‐bp tiles for both wt Col and ddm1. Tiles were categorized by annotation feature.
B Of the tiles in (A) in both wt Col and ddm1, we determined the number of tiles with ≥ 2‐fold CHH methylation dependent on RDR6.
C Distribution of AGO6 enrichment across TEs (solid lines) and genes (dashed lines) for different sizes of small RNAs in either the wt Col TE‐silenced epigenome or the ddm1 TE‐transcriptionally active epigenome. Dashed lines have the same color codes as the solid lines. ‘Start’ and ‘Stop’ represent the edges of the TE and the transcriptional start and stop sites of the genes.
D–G Heatmaps of AGO6 enrichment of TE siRNA size classes in wt Col and ddm1 epigenomes. Analyzed were (D) the position of the TE on a chromosome and TE size, (E) the copy number of the TE, (F) the TE type and (G) the TE family name. The color of the TE family name in (G) corresponds to the TE type in (F).
H Bisulfite sequencing of TE targets. Error bars represent Wilson score interval 95% confidence limits.
ARGONAUTE 6 bridges transposable element mRNA‐derived siRNAs to the establishment of DNA methylation (2014)
Figure 5

RDR6‐RdDM is tissue specific

A Bisulfite sequencing of the Athila6A TSS in juvenile leaf tissue.
B qRT–PCR analysis of Athila6A gag/pol steady‐state mRNA transcript levels in inflorescence and leaf tissue.
C qRT–PCR analysis of AGO6 and AGO4 mRNA transcript levels in inflorescence and juvenile leaf tissue.
D Western blot analysis of wt Col leaf and inflorescence. The arrow denotes the AGO6 protein; the higher band is a previously observed cross‐reactive band (see also Supplementary Fig S3A).
E, F Side view of an inflorescence with (E) or without (F) the AGO6‐GFP transgene.
G, H Top‐down view of an inflorescence with (G) or without (H) the AGO6‐GFP transgene.
I Magnified view of a stage 4–5 flower bud carrying the AGO6‐GFP transgene.
J Nuclear and cytoplasmic fractionation of inflorescence protein. The cytoplasm‐enriched protein represents 2.5% of the total nuclear‐enriched protein. H3Ac is a nuclear protein marker, while PEPC is a cytoplasmic protein.
K CHH methylation levels of the RDR6‐RdDM targets TAS3a and Athila6A TSS in whole‐inflorescence‐tissue samples compared to dissected young buds corresponding to floral stages 6–8.

Data information: In (B) and (C), data are represented as mean ± SD of three biological replicates for each genotype, and differences were analyzed with a two‐tailed unpaired Student's t‐test. In (E–I), auto‐fluorescence of chlorophyll is shown in red. Infl, inflorescence.
PECAM-1 supports leukocyte diapedesis by tension-dependent dephosphorylation of VE-cadherin (2021)
Figure 1

Figure 1. SHP2 supports leukocyte diapedesis by directly dephosphorylating VE-cadherin-Y731.

A Transmigration of mouse neutrophils towards the chemokine CXCL-1 through TNF-α-stimulated VE-cadherin WT (WT) or VE-cadherin Y731F (Y731F) primary endothelial cells, each transfected with control or SHP2-specific siRNA. The transmigration rate is presented relative to that of WT cells transfected with control siRNA, set as 100%. On the right, Western blot analysis of lysates from cells used in transmigration assays for the expression of SHP2 and α-tubulin.

B Transmigration of human neutrophils towards the chemokine IL-8 through TNFα-stimulated human endothelial cells (HUVEC), transfected with control or SHP2-specific siRNA and were either not transduced (-) or transduced (+) with adenoviruses expressing human SHP2 (Adv-SHP2). The transmigration rate is presented relative to that of cells transfected with control siRNA, set as 100%. On the right, Western blot analysis of lysates from cells used in transmigration assays for the expression of SHP2 and α-tubulin.

C Lysates of WT or Y731F primary endothelial cells were submitted to pulldowns with GST, GST‐SHP2‐WT, or GST‐SHP2‐DA. Pulldowns and total cell lysates were analyzed by Western blotting for VE-cadherin and GST. Graph below represents quantification of two independent experiments, presented as fold change of bound VE-cadherin.

D TNFα-activated bEnd.5 cells were incubated with mouse T cells for 0-30 min. SHP2 (above) or VE-cadherin (below) were immunoprecipitated (IP) from cell lysates and precipitates as well as total cell lysates were analyzed by immunoblotting for indicated antigens. Molecular weight markers are indicated in kDa. Graph below represents quantification of five independent experiments, presented as fold change of bound VE-cadherin.

Data information: Immunoblots are representative of two (C) or five (D) independent experiments and transmigration experiments represent data from four (A) and three (B) independent experiments with 3-6 values for each group per assay. Graphs represent mean ± SEM. Statistical significance was tested with one‐way ANOVA, (A, B) or Kruskal-Wallis test (D), *P<0.05, **P <0.01, ****P <0.0001, n.s., not significant.
PECAM-1 supports leukocyte diapedesis by tension-dependent dephosphorylation of VE-cadherin (2021)
Figure 2

Figure 2. PECAM-1 provides SHP2 and is required for leukocyte-induced dephosphorylation of VE-cadherin-Y731.

A - E Different endothelial cells were stimulated with TNF-α for 17 h prior to adding leukocytes for indicated time points followed by immunoprecipitations (antigens indicated above) and immunoblots (antigens indicated on the right) of precipitates and endothelial cell lysates (Iso = isotype control). (A) bEnd.5 cells were incubated with antigen-stimulated mouse T cells for 0-15 min. (B) luEnd cells devoid of PECAM-1 (KO) or re-transfected with PECAM-1 (RT) were incubated with T cells for 30 min. (C) HUVEC transfected with control or PECAM-1-specific siRNA were incubated with differentiated HL60 cells for 20 min. (D) bEnd.5 cells were pre-treated with anti-PECAM-1 (Mec13.3) or anti-endomucin antibodies for 30 min prior to incubation with T cells for 30 min. (E) HUVEC, siRNA depleted for endogenous PECAM-1, were transduced with wild-type PECAM-1 (PECAM-1 WT) or Y663,686F PECAM-1 double mutant (PECAM-1 YF) and incubated with HL60-derived neutrophils for 20 min. On the right, immunoblot analysis of lysates from siRNA treated cells before PECAM-1 re-expression. F Transmigration of human neutrophils towards IL-8 through TNFα-stimulated HUVEC expressing PECAM-1 WT or PECAM-1-Y663,686F. The transmigration rate is presented relative to that of PECAM-1 WT expressing cells, set as 100%.

Data information: Graphs below the immunoblots represent quantification of three (A, B), two (C, E) or four (D) independent experiments, presented as fold change in pY731-VE-cadherin signal intensities, adjusted to the amount of precipitated VE-cadherin and normalized to control cell, set as 1. Immunoblots are representative of three (A, B) independent experiments and transmigration results from three (F) independent experiments with 6 values for each group per assay (mean ± SEM). Statistical significance was tested with one‐way ANOVA (A, B, D) or unpaired two-tailed t-test (F), *P<0.05, **P <0.01, n.s., not significant.
PECAM-1 supports leukocyte diapedesis by tension-dependent dephosphorylation of VE-cadherin (2021)
Figure 3

Figure 3. PECAM-1 is required for leukocyte-induced internalization of VE-cadherin.

A, B Endocytosis of VE-cadherin in 17 h TNF-α treated HUVEC was induced by adding HL60-derived neutrophils for 20-30 min at 37°C and monitored by the internalization of preincubated mAb BV6, which was visualized in fixed cells with a secondary fluorescent antibody (white). Total VE-cadherin was stained with another mAb (red), nuclei stained with Hoechst (white). (A) HUVEC had been transfected prior to the experiment with control or PECAM-1-specific siRNA. Cell lysates were analyzed for expression levels of PECAM-1 and tubulin (below). (B) Similar as A, except that PECAM-1 siRNA treated HUVEC were transduced with WT PECAM-1-EGFP or PECAM-1-Y663,686F-EGFP. Expression levels of transduced PECAM-1 forms and of EGFP and VE-cadherin are shown in immunoblots (on the right). Relative VE-cadherin internalization efficiency is given on the right.

Data information: Graphs represent data from three independent experiments (mean ±SEM) (A, B). Statistical significance was tested with one-way ANOVA, ****P < 0.0001, n.s., not significant. Scale bars 20 μm.
PECAM-1 supports leukocyte diapedesis by tension-dependent dephosphorylation of VE-cadherin (2021)
Figure 4

Figure 4. PECAM-1 mediated leukocyte diapedesis is in vivo and in vitro dependent on VE-cadherin-Y731.

A Transmigration of mouse neutrophils towards CXCL-1 through TNF-α-stimulated bEnd.5 cells transfected with control or SHP2-specific siRNA and pre-treated with anti-endomucin (7C7.1) or anti-PECAM-1 (Mec13.3) antibodies for 30 min at 37°C before addition of neutrophils. The transmigration rate is presented relative to that of control cells, set as 100%.

B Transmigration of mouse neutrophils towards CXCL-1 through TNF-α-stimulated WT or Y731F primary mouse endothelial cells pre-treated with isotype-control or anti-PECAM-1 (2H8) antibodies for 30 min at 37°C prior to addition of neutrophils. The transmigration rate is presented relative to that of control cells, set as 100%.

C - E Extravasated leukocytes (C), adherent leukocytes (D) and rolling flux fraction of leukocytes (E) in cremaster tissue from WT or Y731F VE-cadherin mice stimulated intrascrotally with IL-1β and treated i.v. with isotype-control or anti-PECAM-1 (2H8) antibodies for 4 h before intravital microscopy.

Data information: Graphs represent data from three (A, B) independent experiments in triplicate for each group (mean ± SEM). Data in C - E are of 38 vessels from four mice, 39 vessels from four mice, 31 vessels from three mice and 43 vessels from four mice analyzed in WT+IgG, WT+2H8, Y731F+IgG and Y731F+2H8 treatment groups, respectively (mean ± SEM). Statistical significance was tested with one‐way ANOVA, *P <0.05, **P <0.01, ****P <0.0001, n.s., not significant.
PECAM-1 supports leukocyte diapedesis by tension-dependent dephosphorylation of VE-cadherin (2021)
Figure 5

Figure 5. VE-cadherin-Y731 becomes phosphorylated before it reaches the cell surface and is masked by catenins.

A HUVEC were transduced with increasing titers of adenovirus expressing human SHP2. VE-cadherin was immunoprecipitated from cell lysates and precipitates as well as total cell lysates were analyzed by immunoblots for indicated antigens.

B bEnd.3 cells were lysed and VE-cadherin or immature VE-cadherin was immunoprecipitated respectively with C5 or VD47 antibodies. Precipitates were blotted against pY731-VE-cadherin, β -catenin, plakoglobin, p120, α -catenin and VE-cadherin.

C VE-cadherin-catenin complex was immunoprecipitated from HUVEC lysates and was either left untreated (no SDS) or treated with 0.2% SDS for 30 min at room temperature, followed by removal of SDS and addition of 2 μg recombinant human SHP2 for 30 min at 30°C. Precipitates were analyzed in immunoblots for pY731-VE-cadherin, plakoglobin, β-catenin and VE-cadherin.

D Quantification of pY731 blot signals of three independent experiments (from C), with the signal intensity for untreated VE-cadherin set as 1. Molecular weight markers are indicated in kDa.

Data information: Immunoblots are representative of at least three (A, B-right panel, C) or five (B-left panel) independent experiments (mean ± SEM). Statistical significance was tested with unpaired two-tailed t-test, *P <0.05, n.s., not significant.
PECAM-1 supports leukocyte diapedesis by tension-dependent dephosphorylation of VE-cadherin (2021)
Figure 6

Figure 6. Dephosphorylation of VE-cadherin-Y731 requires Ca2+ mobilization and actomyosin contraction during leukocyte diapedesis.

A Transmigration of mouse neutrophils towards the chemokine CXCL-1 through TNFα-stimulated WT or Y731F primary endothelial cells, either pre-treated with vehicle control (DMSO) or Ca2+ chelator (MAPTAM) for 30 min at 37°C prior to addition of neutrophils. The transmigration rate is presented relative to DMSO-treated endothelial cells expressing WT VE-cadherin.

B TNFα-activated bEnd.5 cells were treated with DMSO or MAPTAM, prior to incubation with antigen-stimulated mouse T cells for 30 min. VE-cadherin was immunoprecipitated from cell lysates and precipitates as well as total cell lysates were analyzed by immunoblots for indicated antigens. Quantification of pY731 blot signals of five independent experiments are shown below, with the signal intensity for the control sample (no T cells, vehicle-control) set as 100%.

C Transmigration assays as in A, except for replacing MAPTAM treatment with Blebbistatin.

D Similar as for B, except for replacing MAPTAM treatment with Blebbistatin.

E HUVEC transduced with lacZ or with SHP2 were either untreated (-) or treated (+) with thrombin for 30 min followed by immunoblotting either VE-cadherin immunoprecipitates or cell lysates for the indicated antigens on the right. A quantification of four independent experiments is shown on the right. Molecular weight markers are indicated in kDa. The immunoblots are representative of five (B) or four (D, E) independent experiments and the quantifications of transmigration experiments represent data from three (A, C) independent experiments in triplicate for each group per assay. Bars and error bars indicate mean ± SEM. Statistical significance was tested with one-way ANOVA (B, E) or 2way ANOVA (A, C), ***P <0.001, ****P <0.0001, n.s., not significant.
PECAM-1 supports leukocyte diapedesis by tension-dependent dephosphorylation of VE-cadherin (2021)
Figure 7

Figure 7. VE-cadherin tension sensor is suitable to measure induction of force across VE-cadherin during thrombin stimulation.
A Schematic representation of VE- cadherin tension sensor (VE-cad FL) and VE-cadherin tailless sensor (VE-cad FL-TS). VE- cadherin tension sensor has a ferredoxin-like (FL) linker-based FRET module inserted within VE-cadherin between the p120 and the β-catenin-binding site, whereas VE-cadherin tailless sensor lacks the catenin binding region of VE-cadherin. The FRET module (depicted in box) consists of two fluorophores, YPet and mCherry separated by an elastic domain. When the module is under tension, FRET from YPet to mCherry decreases.

B HUVEC were transfected with control or VE-cadherin-specific siRNA and transduced with VE-cad FL or VE-cad FL-TS. Cell lysates were immunoblotted for VE-cadherin and actin.

C Immunofluorescence staining for VE-cadherin of HUVEC transfected with control or VE‑cadherin specific siRNA (VE-cad kd) and transduced with VE-cad FL or VE-cad FL-TS. D HUVEC were either transfected with control or VE-cadherin targeting siRNA and transduced with VE‑cad FL or VE-cad FL‑TS. The tension sensor was immunoprecipitated from cell lysates using an anti-GFP antibody. Precipitates as well as total cell lysates were analyzed by immunoblotting for indicated antigens. Molecular weight markers are indicated in kDa.

E, Quantification of FRET efficiency (percentage) in junctions of HUVEC expressing VE-cad FL or VE-cad FL-TS, after thrombin stimulation (1 U/ml) or under control conditions.

Data information: (C, D) Representative pictures from three independent experiments. (E) Graph represents measurements (n = 60, 61, 52, 53) pooled from three independent experiments. Statistical significance was tested with unpaired t-test, ****P<0.0001, n.s., not significant. Scale bars 20 µm.