EMBO/soda-nel huggingface dataset:
Distinct roles of VE-cadherin for development and maintenance of specific lymph vessel beds (2018)
Figure 8

Fig. 8: Enhanced collective cell migration in VE-cadherin-deficient lymphatic endothelial cells.

(A) Collective cell migration of VE-cadherin-deficient and control LECs was assessed in wound scratch assays. LECs were grown up to four passages and seeded at a density of 5x104 cells on fibronectin-coated 8 well µ-chamber slides (growth area 0.22 cm2); scale bar 500 µm

(B) The area of the zone denuded from cells is depicted in the curve diagram showing mean values ± SD of three individual experiments conducted in quadruplets. * p ≤ 0.01. Two-tailed unpaired Student's t test.

Expanded View Figures - Legends
A single epidermal stem cell strategy for safe ex vivo gene therapy. (2015)
Figure 3

Isolation of genetically corrected recessive dystrophic epidermolysis bullosa (RDEB) epidermal stem cells

Single cells were isolated from a mass culture (passage V) of RDEB keratinocytes infected with SIN retroviruses bearing a COL7A1 cDNA. Clonal types were determined (Barrandon & Green, 1987) and listed in Supplementary Table S1. Growing clones were expanded for further characterisation.

COLVII detection in clones by immunostaining. COLVII expression (green) was detectable in some clones (6, 17, 22, 58 and 61) and not in others (3, 24 and 54); nuclei were stained with Hoechst 33342 (blue). Dotted lines delimit the periphery of keratinocyte colonies from the surrounding irradiated 3T3-J2 feeder cells. Scale bar: 50 μm.

Quantitative RT–PCR analysis of COL7A1 expression in transduced clones compared to untransduced RDEB keratinocytes. All clones shown in (A) were transduced but expressed different levels of COL7A1 transcripts. Clones 6, 17, 22, 54, 58 and 61 expressed higher levels of COL7A1 than control RDEB cells and keratinocytes obtained from healthy donors (YF29 and OR-CA, control 1 and 2, respectively). The level of COL7A1 expression in the RDEB untransduced cells was referenced as 1.

Determination of proviral rearrangements in transduced clones. A Southern blot was performed using genomic DNA of RDEB cells, clones and the infected mass culture from which the clones were isolated. Genomic DNA was digested with EcoRV and SpeI that cut at the 3′ and 5′ end of the provirus (Supplementary Fig S2) and hybridised with a 907-bp COL7A1 probe radiolabelled with 32P isotope. The upper band corresponded to the endogenous signal. The retroviral producer line Flp293A-E1aColVII1 was used as a control for the digested 9.6-kb provirus (proviral signal). Smaller bands corresponded to rearranged proviruses marked with an asterisk.

Identification of stem cells producing COLVII. Western blotting revealed that only clone 6 secreted COLVII in the culture supernatant, while clone 54 and surprisingly clone 22 did not (see A). RDEB cells were used as a negative control and healthy donor cells as a positive control. The secreted matrix metalloproteinase 2 (MMP2) was used as a loading control.
A single epidermal stem cell strategy for safe ex vivo gene therapy. (2015)
Figure 4

Long-term restoration of COLVII expression, generation of epidermis and anchoring fibrils by the progeny of a corrected recessive dystrophic epidermolysis bullosa (RDEB) epidermal stem cell

Serial cultivation of transduced and untransduced holoclones demonstrated that the growth potential of the stem cells was not affected by the production of COLVII. Non-COLVII-producing holoclone 54 (black lines) and COLVII-producing holoclone 6 (red lines) were serially transferred once a week until exhaustion (Rochat et al, 1994).

Theoretical number of epidermal cells available for characterisation and CEA production from corrected (clone 6) and uncorrected stem cells (clone 54) calculated from the day of cloning. The colony-forming efficiency and the percentage of growing colonies for each passage were used to calculate the population doubling, the generation number and the total progeny of isolated stem cells. Both corrected and uncorrected epidermal stem cells show high growth potential in vitro.

Immunodeficient SCID mice were transplanted with untransduced cultured RDEB keratinocytes (left) or the COLVII-secreting holoclone (clone 6) (right). Punch biopsies were obtained at various times post-transplantation (PT), stained with haematoxylin/eosine (H/E), for human leucocyte antigen-1 (HLA-1) (green) and human COLVII (red). RDEB keratinocytes generated a HLA-1-positive epidermis that adhered poorly to the dermo-epidermal junction (DEJ) (arrow indicates a blister) and absence of COLVII (dotted line delimits the dermis), whereas the corrected keratinocytes produced an epidermis that adhered to the dermis and deposited COLVII (red) at the DEJ. Note the presence of KI67-positive keratinocytes (red) in the basal and suprabasal layers in the corrected epidermis at 385 days post-transplantation, indicating that transplanted cells had self-renewed for more than a year. Scale bar: 50 μm.

Transmission electron microscopy (TEM) demonstrated the presence of anchoring fibrils (arrows) at the DEJ of the corrected epidermis (middle right panel) and in a normal human skin biopsy (left panel). Note the absence of anchoring fibrils in the RDEB epidermis (middle left panel). Detection of human COLVII by immunogold staining in the DEJ of corrected epidermis (right panel). Scale bar: 250 nm.
A single epidermal stem cell strategy for safe ex vivo gene therapy. (2015)
Figure 5

Characterisation of proviral integrations in corrected stem cells

Visualisation of COL7A1 sequence by FISH analysis in corrected stem cells at passage XII (clone 6). Five specific signals (red) were detected on five different chromosomes. As expected, a COL7A1 probe (red) hybridised to the two endogenous COL7A1 alleles located on the chromosomes 3 as identified by a specific centromeric probe (Cen 3) and to three other chromosomes identified as chromosomes 2, 11 and 22 by means of specific centromeric probes (Cen 2, Cen 11 and Cen 22, respectively). The latter corresponded to proviruses.

Determination of proviral integration sites in corrected stem cell. Genomic DNA from clone 6 was submitted to whole-genome sequencing together with PCR and subsequent Sanger sequencing to identify the integration sites to the base-pair level. Mate pairs that span from the viral sequence to a human chromosome were extracted and used to estimate integration regions. The reads were mapped to the hg19 reference sequence. Three integration sites were uncovered: one in chromosome 2, one in chromosome 11 and one in chromosome 22, and targeted genes were identified.

Analysis of the level of expression of targeted genes in corrected stem cells by quantitative RT–PCR. DARS was not significantly changed in clone 6 compared to the cells before transduction. Primers used annealed downstream of the proviral integration site. Error bars represent the standard deviation of three replicates.

Identification of sequence abnormalities in the two alleles of the DARS-targeted gene in clone 6 by NGS. Reads were mapped to the hg19 reference sequence. Small insertion–deletions and SNP calling were performed and compared to GWAS databases. The mapping highlighted two indels and one SNP in the DARS genomic sequence. The indels were not associated with disease and the SNP was synonymous (CCU–CCG both codons correspond to proline).
A single epidermal stem cell strategy for safe ex vivo gene therapy. (2015)
Figure 6

Assessment of immortalisation, tumorigenicity and disseminative potential of the corrected stem cells

Upper panel: expression of proteins associated with the acquisition of immortalisation process involved in senescence, in evasion of growth suppression and in apoptosis (Hanahan & Weinberg, 2011). The level of P53, P21, RAS and P16 was similar in clone 6, untransduced cells and mass culture from four independent infections, significantly different from the squamous cell carcinoma cell line SCC-13. Lower panel: the phosphorylation state of the PRB restriction point was maintained in clone 6 and untransduced recessive dystrophic epidermolysis bullosa (RDEB) cells, whereas PRB was heavily phosphorylated in transformed cells (SCC-13). Appropriate loading controls were used for each cellular extract (GAPDH for cytoplasmic extracts, histone H3 for nuclear extracts and tubulin for whole-cell extracts. H3 for histone H3, ppRB for hyperphosphorylated pRB and pRB for hypophosphorylated PRB).

Transduced clone 6 had a diploid karyotype at passage XVI (see also Fig5A).

Transduced clones were not tumorigenic. The tumorigenic potential of the clones was tested by tumour formation in nude mice. Transduced RDEB holoclones (blue) [clone 6 (square, n = 4) passage X, clone 22 (circle, n = 2) passage XI, clone 54 (cross, n = 4) passage X] were not tumorigenic when injected subcutaneously in athymic mice as were untransduced RDEB keratinocytes (passage VII). SCC-13, a squamous cell carcinoma cell line (black) (lozenge, n = 2), was used as a positive control.

To test whether the transduced RDEB keratinocytes had disseminated after the generation of an epidermis onto SCID mice (see Fig4C), the internal organs of the recipient mice (3 mice for clone 6 and 2 mice for clone 22) were harvested 385 days post-transplantation and analysed for COL7A1 proviral sequences. No COL7A1 sequence was detected. A mouse transplanted with a holoclone obtained from a healthy donor (YF29) was used as an internal control. PCR-positive controls were genomic DNA from transduced cells (holoclone 6) and cDNAs isolated from healthy keratinocytes. B-actin was run as a loading control.
Individual cristae within the same mitochondrion display different membrane potentials and are functionally independent. (2019)
Figure 1

Figure 1. High-resolution fluorescence imaging using Airyscan resolves the inner mitochondrial membrane (IMM) structure in live cells.

A-F. High-resolution imaging of mitochondria in live cells using the Airyscan module of Zeiss LSM880 confocal microscope.

A. Images of IMM in living HeLa, L6, and H1975 cells, stained with 10-N-nonyl acridine orange (NAO). NAO preferentially binds phospholipids in the IMM, such as cardiolipin. Arrowheads indicate cristae in the IMM. Scale bar = 500 nm. N ≥ 3 independent experiments for each cell type.

B. Mitochondrion cropped from HeLa cell shown in A (dashed line) and zoomed in to show cristae, inner boundary membrane (IBM), and matrix (arrows). Scale bar = 100 nm.

C. H1975 cells transduced with matrix-targeted-DsRed and stained with NAO. Matrix-targeted Ds-Red differentiates matrix from cristae stained with NAO. Arrowheads point to cristae. Scale bar = 500 nm. N = 1 independent experiment.

D. The structure of the IMM in Mic13-KO cells (HeLa), stained with NAO. The number of cristae is decreased in Mic13-KO compared to control cells shown in panel A, labeled with arrowheads. Scale bar = 500 nm. N = 3 independent experiments.

E-F. Live-cell imaging of the IMM in control and PTPMT1 KD H1975 cells, a model of cardiolipin deficiency.

E. A gallery of mitochondria from various control H1975 cells expressing scrambled shRNA and stained with NAO, showing cristae (arrowheads). Scale bars = 500 nm. N = 3 independent experiments.

F. A gallery of mitochondria from various H1975 cells expressing PTPMT1 shRNA and stained with NAO. Note the derangement of the ultrastructure (arrowheads). Scale bars = 500 nm. N = 3 independent experiments.
Individual cristae within the same mitochondrion display different membrane potentials and are functionally independent. (2019)
Figure 6

Figure 6. Crista junctions (CJ) regulate the difference in ΔΨm between cristae and IBM.

The role of cristae structure as well as CJ formation and sealing on the generation of the difference in ΔΨm between cristae and IBM were studied by disrupting cristae using Mic10-, Mic13- and Mic60-deficient cells. Mic13 and Mic60 support cristae formation. Mic10 is essential for CJ formation, and, in its absence, cristae tend to remain as vesicles detached from IBM. Live-cell Airyscan imaging of TMRE was used in all figure panels and models.

A. Representative images of mitochondria in control (top row) vs. Mic13-KO (bottom row) HeLa cells. Note that TMRE FI in Mic13-KO mitochondria is distributed more evenly along the IMM, so that ΔΨCr-IBM is decreased. Scale bar = 500 nm. N = 3 independent experiments.

B. Quantification of A, showing deletion of Mic13 in HeLa cells results in significant decrease in ΔΨCr-IBM. While Mic13-KO cells show a substantial decrease in cristae number, the loss of cristae does not appear absolute, making it feasible to compare TMRE FI at cristae relative to IBM. N = 3 independent experiments.

C. Representative images of Hap1 control (top row) vs. Mic60 KO (middle row) and Mic10 KO (bottom row); Mic60-KO mitochondria show very few cristae structures. TMRE staining along the IMM is relatively homogeneous, indicating decreased ΔΨCr-IBM. In general, Mic10 KO in Hap1 cells results in decreased TMRE signal intensity at cristae relative to IBM (arrowheads) as compared to control cells. Deletion of Mic10 induces detachment of cristae from IBM (see Figure 8). Except for the detached cristae vesicles, the TMRE staining is homogeneously distributed in Mic10-KO cells, indicating decreased ΔΨCr-IBM. Scale bar = 500 nm. N = 3 independent experiments.

D. Quantification of C, showing Mic60 KO in Hap1 cells results in significant decrease in ΔΨCr-IBM. While Mic60-KO cells show a marked loss of normal cristae structures, depletion of cristae does not appear absolute, making it possible to compare TMRE FI at cristae relative to IBM. N = 3 independent experiments.

E. Quantification of C, showing Mic10 KO in Hap1 cells results in a significant decrease in ΔΨCr-IBM. Note: quantification of ΔΨm at cristae relative to IBM was performed only on cristae structures that appeared to maintain attachment to IBM. N = 3 independent experiments.

Data information: Data were analyzed with 2-tailed Student's t-tests, and p values < 0.05 were considered statistically significant. Specific p values are indicated in the figure. Error bars indicate SEM.
Individual cristae within the same mitochondrion display different membrane potentials and are functionally independent. (2019)
Figure 7

Figure 7. Opa1 regulates the difference in ΔΨm between cristae and IBM.

Studies show that Opa1 interacts with MICOS complex, promoting closure of CJs. Thus, we tested whether Opa1 was required to maintain the difference in ΔΨm between cristae and IBM.

A. Live-cell Airyscan images of MEF control (top row) vs. Opa1 KO (bottom row), co-stained with NAO and TMRE. Arrowheads indicate decreased intensity of TMRE FI at cristae compared to IBM. Note the more even distribution of TMRE staining, indicating the cristae and IBM are relatively equipotential. Scale bar = 500 nm.

B. Quantification of G, showing Opa1-KO MEFs have significantly decreased ΔΨCr-IBM. N = 3 independent experiments.

Data information: Data were analyzed with 2-tailed Student's t-tests, and p values < 0.05 were considered statistically significant. Specific p values are indicated in the figure. Error bars indicate SEM.
The fungal ligand chitin directly binds TLR2 and triggers inflammation dependent on oligomer size (2018)
Figure 1

Figure 1: Size-dependent recognition of defined chitin oligomers in humans, mice and plants.

(A) Electron micrograph of a human MoMac engulfing a crude chitin particle (n=2).

B-E (B) Size comparison (to scale) of typical TLR ectodomain and a chitin 7-mer (seven NAG subunits). Release of IL-6 and/or TNF release (ELISA) from (C) human MoMacs (n=6-8), (D) murine BMDMs (n=6) or (E) THP-1 cells (n=3).

(f) GFP in vivo fluorescence post intradermal injection of chitin oligomers into LysMEGFP/+ C57BL/6 mice (n=3/group).

G-K (G) Representative in vivo imaging results. Leukocyte infiltration in vivo after intratracheal administration assessed by (H) BALF flow cytometry (n=3-4/group) and (I) histology analysis (n=2), scale bar = 200 μm, arrows show cellular infiltrate. A. thaliana seedling (J) target gene mRNA induction (n=3) or leaf piece (K) ROS production (0-45 min, n=6).

Data information: (C, D, F, H and K) represent data (mean+SEM) combined from 'n' (given in brackets for each panel) technical or biological replicates (human donors, mice or plant leaves, respectively). In (A, E, G, I and J) one representative of 'n' (given in brackets for each panel) independent experiments is shown (mean+SD). * p<0.05 according to Wilcoxon signed rank sum (C, D), one-way ANOVA (H) or Student's t-test (E, J).
The fungal ligand chitin directly binds TLR2 and triggers inflammation dependent on oligomer size (2018)
Figure 2

Figure 2: TLR2 is the mediator of chitin oligomer-mediated immune activation.

A, B TNF released from WT or Myd88 (A) or Tlr2 (B) KO BMDM (total n=6-9/group combined from 3 experiments).

(C) IL-6 released from primary MoMacs (n=5) treated with non-targeting (NT), TLR2- or MyD88-specific siRNA relative to NT condition.

(D) CD62L shedding from human PMNs (n=4-8) with or without isotype control or anti-TLR2 blocking antibody.

(E) IL-6 protein released from mock or TLR2 Cas9-CRISPR-edited THP-1 cells (n=3).

(F) NF-κB activity in empty vector (EV) or TLR2-transfected HEK293T cells (n=3).

(G) MPO release from intradermally injected WT or Tlr2 KO mice (n=5-15 biopsies each).

(H) BALF IL-6 and TNF from intratracheally treated WT or Tlr2 KO mice (n=4-5 mice each).

(I) Relative IL10 mRNA in whole blood of WT or heterozygous TLR2 R753Q carriers (n=5 each).

Data information: In (A-D, G-I) represent data (mean+SEM) combined from 'n' technical or biological replicates (human donors, mice or samples, respectively). In (E and F) one representative of 'n' independent experiments is shown (mean+SD). * p<0.05 according to Mann-Whitney U (D, I), one-sample t-test (C), Student's t-test (A, B, E-G) or one-way ANOVA with Dunnett's multiple comparison (H).
The fungal ligand chitin directly binds TLR2 and triggers inflammation dependent on oligomer size (2018)
Figure 3

Figure 3: Chitin is a unique fungal TLR2 ligand.

(A) NF-κB activity in TLR2-transfected HEK293T cells (n=3).

(B) IL-8 release and ROS production from primary PMNs (n=7).

(C) NF-κB activity in hTLR2 HEK-Dual cells pretreated with TLR1, TLR2 or TLR6 blocking Abs (n=2).

(D) IRAK4 S186 and T345 phospho-site containing IRAK4 peptides quantified in proteome-wide phospho-screen in THP-1 cells (n=1).

(E) Principal component analysis of microarrays conducted on stimulated whole blood samples from healthy donors (n=3-5).

(F) Microarray intensities (excerpt) of chitin-specific signature genes (n=5).

(G) Relative cytokine mRNAs in whole blood stimulation (n=5-13).

Data information: (B, E-G) represent data (mean+SEM) from 'n' technical or biological replicates (donors). In (A, C and D) one representative of 'n' independent experiments is shown (mean+SD). * p<0.05 according to Wilcoxon signed rank sum (B, G), one-way ANOVA with Sidak correction (C), and Student's t-test (A); for details on data acquisition and analysis for panels (D-F) see Expanded View Information.
The fungal ligand chitin directly binds TLR2 and triggers inflammation dependent on oligomer size (2018)
Figure 4

Figure 4: Direct chitin TLR2 binding and immune activation can be blocked by SSL3 or short chitin oligomers.

A, B Flow cytometric quantification (A) or microscale thermophoresis (MST) analysis (B) of Alexa647-labeled C10-15 interaction with mTLR2-Fc Protein (n=4 each).

C, D (C) Flow cytometry and (D) fluorescence microscopy of Candida albicans stained with control IgG or mTLR2-Fc anti-Fc-Alexa594, together with ConA-Alexa488 and CFW (n=4 each). Scale bar = 5 μm.

E, F (E) Docking of chitin 10 (magenta) into the TLR2 (orange) Pam2 (green) lipopeptide binding pocket (close-up, pdb: 2z7x), (F) overlayed with SSL3 from a SSL3-inhibited TLR2 complex (pdb: 5d3i).

(g) MST analysis of Alexa647-labeled C10-15 and mTLR2-Fc protein with SSL3 titration (n=2).

H, I (H) NF-κB activation in TLR2-transfected HEK293T cells (n=3) or (I) IL-8 release from primary PMNs (n=2) without or with SSL3.

(J) BALF TNF in C57BL/6 mice (n=5/group) upon C10-15 administration without or with SSL3.

(K) ROS production between 0 and 45 min post chitin application in A. thaliana leaf pieces (n=6).

(L) MST analysis of Alexa647-labeled C10-15 and mTLR2-Fc Protein with C5 titration (n=2).

Data information: Quantification sub-panels (Aand C) and panels (K and J) represent data (mean+SEM in A, C and J; mean±SD in K) combined from 'n' biological replicates (human donors, mice or plant leaves, respectively). In (A-D, G-I and L) one representative of 'n' independent experiments is shown (for H and I asmean+SD). * p<0.05 according to Mann-Whitney U (A), one-sample t-test (C), Student's t-test (H, I) or one-way ANOVA with Dunnett's multiple comparison (J).

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Integrated Genomics of Crohn's Disease Risk Variant Identifies a Role for CLEC12A in Antibacterial Autophagy. (2015)
Figure 1

Perturbational Profiling Reveals Genotype-Specific Transcriptional Responses, Including Differentially Regulated Genes that Are Required for Autophagy

(A) Gene expression analysis of PBMCs isolated from healthy individuals after infection or stimulation with Borrelia burgdorferi (Bor), MDP, or Pam3Cys (Pam). Heatmap shows log2 (fold change) relative to mock-treated control.

(B) Heatmap identifying transcripts exhibiting response differences to infection or immune stimuli, stratified by ATG16L1 T300A polymorphism status.

(C) HeLa GFP-LC3 cells were transfected with indicated siRNAs and infected with DsRed-labeled Salmonella. The percentage of GFP-LC3+ Salmonella at 1 hr post-infection is shown. Data are shown as means ± SD; n = 75 cells per condition from three biological replicates. Data are representative of three independent experiments.

(D) Representative epifluorescent microscopy images of HeLa cells used to generate data shown in (C). The scale bars represent 10 μm.

(E) Western blot of HeLa cells transfected with the indicated siRNAs and treated with Torin E64D/PepA. Data are representative of three independent experiments.

(F) CLEC12A rescue constructs indicating positions of synonymous mutations and location of targeting siRNA.

(G) HeLa GFP-LC3 cells were transfected with control siRNA, CLEC12A siRNA1, or CLEC12A siRNA2 as well as CLEC12A rescue constructs (R1 or R2) and infected for 1 hr with DsRed-labeled Salmonella. Data are shown as mean ± SD; n = 75 per condition. Data are representative of three independent experiments.

(H) HeLa cells from (G) were lysed, and HA-CLEC12A expression levels were assessed by western blot. Data are representative of three independent experiments.

See also Figure S1.
Integrated Genomics of Crohn's Disease Risk Variant Identifies a Role for CLEC12A in Antibacterial Autophagy. (2015)
Figure 2

CLEC12A Knockdown and the ATG16L1 T300A SNP Act Additively to Impair LC3-Bacteria Colocalization

(A) Representative epifluorescent images showing impaired antibacterial autophagy in ATG16L1 KO HeLa cells complemented with the indicated ATG16L1 protein in the presence of control siRNA or CLEC12A siRNA. The scale bars represent 10 μm.

(B) Percentage of LC3-Salmonella colocalization in WT HeLa cells, ATG16L1 KO HeLa cells, and ATG16L1 KO HeLa cells stably expressing ATG16L1∗300T or ∗300A alleles in the presence of control siRNA or CLEC12A siRNA. Data are shown as means ± SD; n = 75. Data shown are representative of three independent experiments.

See also Figure S2.