EMBO/soda-nel huggingface dataset:
Porphyromonas gingivalis evasion of autophagy and intracellular killing by human myeloid dendritic cells involves DC-SIGN-TLR2 crosstalk. (2015)
Fig 8

TLRs activation restores LC3-II expression and inhibits the growth of Mfa1<sup>+</sup>Pg within human MoDCs.

A) Flow cytometry of CD83 on MoDCs after incubation of TLR4 ligand (E. coli LPS) and TLR1 and 2 ligand (Pam3csk4) for 4 hour. B) Immuno-fluorescence images of LC3-II (red) within MoDCs after incubation with TLR4 and TLR1&2 ligands (E. coli LPS and Pam3csk4) C) The plot represents the means ±standard deviation of CFU within MoDCs harvested from three healthy individuals after 24 hours (** p<0.001).
Porphyromonas gingivalis evasion of autophagy and intracellular killing by human myeloid dendritic cells involves DC-SIGN-TLR2 crosstalk. (2015)
Fig 9

Formation of double-membrane vesicles in <italic>P. gingivalis</italic>-infected MoDCs.

A) Scanning electron microscopy (SEM) of MoDCs of the early interaction of MoDCs with Pg381 and Mfa1+Pg (upper panel 10000x and lower panel 25000x). B) SEM for MoDCs interacting with Pg381 (green stains for bacteria are computer generated). C) Transmission electron microscopy (TEM) of autophagosome like structures within MoDCs infected with Pg381, Mfa1+Pg and FimA+Pg for 12 hours (upper, middle and lower panels, respectively). The right and left sections show the different magnifications of randomly selected sections. Pg381 and FimA+Pg strains are mostly enclosed in the characteristics double-membrane intracellular vesicles (Orange arrows). Contrary, Mfa1+Pg escaped these autophagic (double-membrane) vesicles and enclosed within single membrane structures or freely occupy the cytoplasm (Green arrows). D) The ratio of bacteria included in the double membrane were compared to total number of bacteria within MoDCs and plotted as percentage. Counting of the bacteria included in single or doubled membrane vesicles after 12 hours of infection. Each strain was counted in three randomly selected grids for each sample. The analysis of the bacterial counts used Kruskal-Wallis test of different groups and Dunn’s test for multiple comparisons (*p<0.01).
Porphyromonas gingivalis evasion of autophagy and intracellular killing by human myeloid dendritic cells involves DC-SIGN-TLR2 crosstalk. (2015)
Fig 10

LAMP1 decreased within MoDCs infected with <italic>P. gingivalis</italic> expressing Mfa1.

A) Epifluorescence microscopy images of MoDCs infected with pre-labeled bacteria (green-CFSE). LAMP1 detected by transduction of red fluorescent protein (RFP) chimera using baculovirus transgenes to MoDCs. B) The mean of fluorescent intensities ±standard deviation of LAMP1 were quantified in three different experiments (* p<0.001). C) The upper scattered plot shows the co-localization of red and green signals in sections of MoDCs infected with Pg381 and mutants strains. The lower plot shows the average of Pearson’s correlation measures of three randomly selected region of interest (ROI) within each field within 3 different experiments (* p<0.0001) (Table 4 and 5). All analysis of fluorescence intensity used One-way ANOVA analysis of different groups and Tukey’s test for multiple comparisons.
Targeting DDR1 and DDR2 overcomes matrix-mediated melanoma cell adaptation to BRAF-targeted therapy (2021)
Figure 1

Figure 1. Composition, topology and mechanical properties of fibroblast-derived 3D ECMs.

A Images show collagen matrix gel contraction by HDF (human dermal fibroblasts), skin-MAF (melanoma associated fibroblasts isolated from skin lesions), LN-FRC (lymph node fibroblast reticular cells), and LN-MAF (melanoma associated fibroblasts isolated from metastatic lymph node). Dashed circles represent the diameter of the gel. Data are representative of n = 3 independent experiments.

B Immunofluorescence analysis of fibronectin (green) and collagen (red) fibers on de-cellularized ECM produced by human fibroblasts. Fibers orientation was quantified using ImageJ software. Percentages indicate oriented fibers accumulated in a range of ± 21° around the modal angle. Data is represented as mean ± s.d. (n=10 random fields from 2 independent determinations)..Scale bar, 50 µm. A representative image of picrosirius red staining from 10 analyzed is shown for each condition.

C Atomic force microscopy (AFM) measurement of the elastic properties (apparent Young's Modulus, Εapp) of fibroblast-derived ECMs. Each dot represents a specific Young's Modulus obtained by fitting the corresponding individual force-curve acquired on a determined point of the sample. A representative experiment from 2 independent experiments is shown. Scatter plots show mean ± SEM. The black bars represent the median and the interquartile range. ****P<0.0001, Two-tailed Mann-Whitney test.
Targeting DDR1 and DDR2 overcomes matrix-mediated melanoma cell adaptation to BRAF-targeted therapy (2021)
Figure 2

Figure 2. Fibroblast-derived 3D ECM confers drug-protective action to melanoma cells against anti-BRAFV600 therapies.

A Scheme of the ECM-mediated drug protection assay.

B Illustration of the BRAFV600E pathway and the MAPK pathway inhibitors used in the study.

C Time-lapse imaging of proliferation of NucLight-labeled 1205Lu cells plated on plastic (left panel), Coll-1 (collagen-1; middle panel) or FRC-derived ECM (right panel) and treated with vehicle, 5 µM BRAFi or 2 µM BRAFi plus 0.01 µM MEKi using the IncuCyte ZOOM system. Each data point represents the mean of NucLight red nuclear objects per field ± SEM. ****P<0.0001, two-way ANOVA followed by Dunnett's multiple comparison test. Data are representative of n = 3 independent experiments.

D, E Quantification of proliferation of 1205Lu (D) and SKMEL5 (E) cells plated for 48 h on plastic, Coll-1 or the indicated fibroblast-derived ECMs prior a 96 h treatment with vehicle, 5 µM BRAFi or 2 µM BRAFi plus 0.01 µM MEKi. Cells were counted by Hoechst-labeled nuclei staining. Data are represented as bar plots with mean ± SEM normalized to vehicle of 3 independent experiments. (D) **P=0.015, ***P=0.0009, ****P<0.0001 and (E) *P=0.0204, ***P=0.0006, ****P<0.0001. Kruskal-Wallis followed by Dunn's multiple comparisons test.

F Flow cytometry analysis of cell cycle distribution of SKMEL5 cells cultured on plastic, Coll-1 or the indicated fibroblast-derived ECMs and treated with vehicle or 2 µM BRAFi combined with 0.01 µM MEKi. The percentage of cells in different phases of the cell cycle is indicated.

G Immunoblotting of protein extracts from SKMEL5 cells cultivated as described above on plastic, Coll-1 or the indicated fibroblast-derived ECMs in the presence or not of BRAFi or BRAFi/MEKi for 96 h, using antibodies against P-ERK1/2, ERK2 or cell cycle markers (P-Rb, Rb, Cyclin D1, p27KIP1, Survivin, p53). HSP60, loading control.
Targeting DDR1 and DDR2 overcomes matrix-mediated melanoma cell adaptation to BRAF-targeted therapy (2021)
Figure 3

Figure 3. Expression of DDR1 and DDR2 in human melanoma.

A Meta-analysis of 363 cutaneous melanoma from TCGA (skin cutaneous melanoma, PanCancer Atlas) (http://www.cbioportal.org/) showing the percentage of samples with genetic alterations of DDR1 and DDR2. Cases with missense (green) and truncating (blue) mutations, amplification (red) and mRNA overexpression (pink) are indicated; gray, individual cases.

B Immunohistochemical analysis of DDR1 and DDR2 levels on human melanoma tissue microarrays. Representative IHC images and quantification (right bar histograms) of DDR1 and DDR2 expression in normal skin, naevus, primary melanoma (PM) and lymph node melanoma metastases (MM). Scale bar, 100 µm. Histological scoring of the samples was performed in blinded fashion. Samples were scored as low, medium or high for DDR1 or DDR2 expression (naevus, n = 12; PM, n = 30; MM, n = 20).

C, D, E Immunoblotting of equal amounts of protein extracts from melanoma cell lines (C), patient-derived short-term cell cultures (D) or isogenic pairs of parental sensitive and BRAFi-resistant cell lines (E) using antibodies against DDR1, DDR2 or markers of the melanoma cell differentiation AXL, MITF or SOX10. ERK2, loading control.

F DDR1 and DDR2 levels increase in dedifferentiated melanoma cells. Box and whisker plots show DDR1, DDR2, MITF, AXL and SOX10 expression among four differentiation melanoma cell states (U, Undifferentiated, n=10; NC, Neural crest-like, n=14; T, Transitory, n=12; M, Melanocytic, n=17) (GSE80829). The expression of AXL, MITF and SOX10 is shown as control markers of cell differentiation. n, number of cell lines representative of each cell state. Central bars represent the median and the whiskers the 10th to 90th percentile of the boxplot. Multiple comparisons were performed using ordinary one-way ANOVA.
Targeting DDR1 and DDR2 overcomes matrix-mediated melanoma cell adaptation to BRAF-targeted therapy (2021)
Figure 4

Figure 4. Inhibition of DDR1 and DDR2 by genetic or pharmacological approaches abrogates ECM-mediated resistance to BRAFV600 pathway inhibition.

A Immunoblotting of protein extracts from 1205Lu cells transfected with a siRNA control (CTRL) or two different sequences of siRNA (#1 and #2) directed against DDR1 or DDR2 alone or in combination prior being cultivated on MAF-derived ECM and treated or not with 5 µM BRAFi for 96 h, using antibodies against the indicated proteins. HSP60, loading control.

B Immunoblotting of protein extracts from SKMEL5 cells transfected with siCTRL or the combination siDDR1#2/siDDR2#2 prior being cultivated on FRC- or MAF-derived ECMs (left and right panels, respectively) and treated with vehicle, 5 µM BRAFi or 2 µM BRAFi plus 0.01 µM MEKi, using antibodies against the indicated proteins. HSP60, loading control.

C Quantification of the time-lapse imaging of the proliferation of NucLight-labeled 1205Lu cells using the IncuCyte ZOOM system. Cells were plated for 48 h on FRC- or MAF-derived ECMs prior a 96 h treatment with vehicle or 5 µM BRAFi in the presence or not of 7 µM Imatinib or 1 µM DDR1-IN-1. The bar plots represent the mean normalized to vehicle of NucLight red nuclear objects per field ± SEM from 3 independent experiments performed in triplicate. ****P<0.0001, two-way ANOVA followed by Sidak's multiple comparison test.

D Immunoblotting of protein extracts from 1205Lu, SKMEL5 and MM099 cells plated for 48 h on FRC-derived ECM prior a 96 h treatment with vehicle or 5 µM BRAFi in the presence or not of Imatinib (7 µM for 1205Lu, 10 µM for SKMEL5 and MM099) or DDR1-IN-1 (1 µM for 1205Lu, 5 µM for SKMEL5, 3 µM for MM099), using antibodies against the indicated proteins. HSP60, loading control.

E Flow cytometry analysis of cell death (Annexin V/PI labeling) in 1205Lu cells plated on FRC-derived ECM and treated as above. Right bar plots show the distribution of cells (% of total) across the different forms of death.
Targeting DDR1 and DDR2 overcomes matrix-mediated melanoma cell adaptation to BRAF-targeted therapy (2021)
Figure 5

Figure 5. Interaction of melanoma cells with 3D-ECMs induces the linear clustering of phosphorylated DDR upon BRAFi/MEKi treatment.

A Representative images of 1205 cells cultivated on collagen-I (Coll-I) or FRC-derived ECM for 48 h prior treatment with vehicle or 5 µM BRAFi or 2 µM BRAFi plus 0.01 µM MEKi for 96 h. Immunofluorescence for Phospho-DDR1 (P(Y792)-DDR1) (red; left panels) and Phospho-DDR1/2 (P(Y796)-DDR1/P(Y740)-DDR2) (red; right panels), F-actin (green) and nuclei (blue) is shown. Enlarged images of P-DDR1 and P-DDR1/2 immunostaining are shown. White arrows indicate P-DDR1 and P-DDR1/2 cell membrane linear clustering. Scale bar, 25 µm (enlarged images: scale bar, 10 µm).

B Quantification of globular versus linear clusters of Phospho-DDR1 (left panels) and Phospho-DDR1/2 (right panels) from immunofluorescence staining shown in (A) using Image J software. Prior the quantification of DDRs clusters a "subtract background" function of ImageJ has been applied to all images. In order to quantify clusters, the IsoData threshold has been used. Clusters with circularity 0.3 to 1 has been defined as "globular" and clusters with circularity 0 to 0.29 has been defined as "linear". Data are from >20 individual cells (n = 3). Error bars reflect mean ± s.d. Values for each treated condition are compared to the vehicle control. 2way ANOVA followed by Dunnett's multiple comparisons test.

C Quantification of globular versus linear clusters of Phospho-DDR1 and Phospho-DDR1/2 from immunofluorescence staining shown in Fig EV4A of 1205Lu cells cultivated on HDF- or MAF-derived ECM and treated with the indicated targeted drugs as described in (A). Data are from >20 individual cells. Error bars reflect mean ± s.d. Values for each treated condition are compared to the vehicle control. 2way ANOVA followed by Dunnett's multiple comparisons test.
Targeting DDR1 and DDR2 overcomes matrix-mediated melanoma cell adaptation to BRAF-targeted therapy (2021)
Figure 6

Figure 6. Targeting DDR by Imatinib sensitizes melanoma tumors to BRAFV600E inhibition.

A Outline of the experimental set up and treatment regimens.

B 1205Lu cells were s.c. inoculated into nude mice and when tumors reached 75 mm3, mice were treated with the indicated mono- or combo-therapy for 30 days. Graphs show tumor growth following treatment by indicated drugs. Data shown are mean ± SEM of tumor volume. Vehicle and Imatinib groups, n = 10 tumors from 5 mice; Vemu and Vemu/Imatinib groups, n = 20 tumors from 10 mice). ****P<0.0001, two-way ANOVA followed by Tukey's multiple comparison.

C Scatter plot graphs showing the tumor weight upon treatment by the indicated mono- or combo-therapy. Error bars show mean ± SEM of tumor weight (n = 10 to 11 tumors from 6 mice per condition). ***P=0.0002, Kruskall-Wallis test followed by Dunn's multiple comparison test; ns, non significant.

D Immunofluorescence staining using anti-cleaved Caspase 3 (red), anti-Ki67 (green) and DAPI in tumor sections of 1205Lu-derived xenografts from (B). Scale bar, 100 µm. A microphotograph of the tumor size in each treated group is shown (representative of n = 5 to 10 mice).

E Kaplan-Meier survival curves of mice treated with vehicle, Imatinib, BRAFi or BRAFi plus Imatinib. Median time to progression was 18, 20, 36 and 48 days, respectively. Log rank (Mantel-Cox) for BRAFi vs BRAFi/Imatinib mesylate. ****P<0.0001 and hazard ratio (log rank): 0.2403 (95% CI of ratio, 0.08123 to 0.7106).

F Mouse body weight was measured at the indicated day. Data shown are mean ± SEM (n = 5 to 10 mice).
Targeting DDR1 and DDR2 overcomes matrix-mediated melanoma cell adaptation to BRAF-targeted therapy (2021)
Figure 8

Figure 8. Targeting the non-canonical NFκB2 pathway overcomes DDR-dependent MMDR to BRAFV600E pathway inhibition.

A Immunoblot analysis of protein extracts from siCTRL- or siDDR1/2-transfected SKMEL5 cells plated on FRC- or MAF-derived ECMs in the presence or not of 5 µM BRAFi, 2 µM BRAFi plus 0.01 µM MEKi for 96 h using antibodies against DDR1, DDR2, P-ERK1/2, ERK2, RelB, p100/p52 and HSP60 as loading control were used.

B Immunoblot analysis of protein extracts from siCTRL- or siDDR1/2-transfected MM099 short-term cultures plated on FRC- or MAF-derived ECMs in the presence or not of 2 µM BRAFi plus 0.01 µM MEKi for 96 h using antibodies as above. Note that data come from the same immunoblot.

C Illustration of the non-canonical p52/RelB NFκB2 pathway and inhibitors used in the study.

D Immunoblot analysis of protein extracts from 1205Lu cells cultivated on plastic, Coll-1 or on indicated fibroblast-derived ECMs for 48 h using antibodies against RelB, p100/p52 and ERK2 as loading control.

E Immunoblot analysis of protein extracts from 1205Lu (left panels) or SKMEL5 (right panels) cells cultivated on FRC-derived ECMs for 96 h in the presence of 5 µM BRAFi and/or a pan-IKK inhibitor (IKKi, BMS-345541 3 µM) using antibodies against P-ERK1/2, P-Rb, Survivin, RelB, and HSP60 as loading control.

F, G Immunoblot analysis of protein extracts obtained from SKMEL5 (F) or MM099 (G) cells plated on FRC- or MAF-derived ECMs and treated with 5 µM BRAFi in combination or not with DDR1-IN-1 (5 µM for SKMEL5 and 3 µM for MM099) or 10 µM NIK inhibitor (NIKi) for 96 h. Antibodies against P-ERK1/2, ERK2, P-Rb, Rb, Survivin, Caspase 3, cleaved Caspase 3, RelB, p100/p52 and HSP60 as loading control were used.
Cyclin O (Ccno) functions during deuterosome‐mediated centriole amplification of multiciliated cells (2015)
Figure 1

Ccno is specifically expressed in multiciliated cells and the embryonic node

A Ccno mRNA is expressed in the embryonic node at E8 as shown by in situ hybridization. Occasionally, additional expression is observed at the posterior tip of the embryo (asterisk). Double labelling by staining for LacZ expression from the CcnoTA allele (shown in B) and in situ hybridization for Dand5, marking crown cells at the circumference of the node. Ccno expression is restricted to ciliated, ventral pit cells of the node as detailed in the transverse section at the indicated plane.
B Schematic of the CcnoTA targeted LacZ knock‐in allele used in (A) and (C–G).
C Whole embryo X‐Gal staining of E16 CcnoTA/+ and wild‐type control embryos and indicated section planes of (D–F').
D–F' Histological sections of LacZ‐stained CcnoTA/+ embryo at E16 reveal Ccno expression in the epithelium of (D) plexus choroideus and ependyme, (E) snout epithelium, and (F, F') trachea and bronchi. pc, plexus choroideus; se, snout epithelium; tr, trachea; and br, bronchus.
G Co‐expression of Ccno and Foxj1 in multiciliated cells of the trachea shown by double staining for X‐Gal and immunohistochemistry using a FOXJ1‐specific antibody.

Data information: Scale bars: 100 μm in (A), 1 mm in (C), 500 μm in (D‐F') and 100 μm in (G).
Cyclin O (Ccno) functions during deuterosome‐mediated centriole amplification of multiciliated cells (2015)
Figure 2

Genetic deletion of Ccno leads to severe hydrocephalus, mucociliary clearance deficits and reduced cilia number of MCCs

A Ccno‐deficient mice develop severe hydrocephalus resulting in characteristic head deformation at P21 (arrow).
B, C Cranial MRI analysis (B) shows the drastically enlarged ventricular cavity (marked by V) and diminished cerebral cortex tissue, (C) also seen when brains are cut in coronal orientation.
D, D' Alcian‐blue staining of paranasal cavities reveals mucus congestion along the nasal epithelium (arrows).
E Survival table of different genotypes at embryonic stages (E13–E17) and at P21 from offspring of heterozygous CcnoRA/+ intercrosses. 11% of CcnoRA/RA homozygous animals die between E17 and P21. Of CcnoRA/RA homozygous animals at P21, roughly 60% develop severe hydrocephalus (20/35 animals), and 40% (14/35) appear grossly unaffected.
F, F' Scanning electron microscopy (SEM) of P21 adult trachea shows reduced numbers of cilia of Ccno‐deficient MCCs. Remaining cilia are found in the central regions of the cell surface, and cell margins frequently lack the ciliary decoration (arrows).
G Transmission electron microscopy (TEM) of wild‐type and CcnoRA/RA MCCs from adult trachea. Ccno‐deficient MCCs show reduced numbers of basal bodies and cilia that correctly docked to the apical cell surface (arrows). Ectopic electron‐dense material is found within the cytoplasm of Ccno‐deficient MCCs (arrowheads).

Data information: Scale bars: 500 μm in (D), 100 μm in (D'), 7 μm in (F), 2 μm in (F') and 1 μm in (G).
Cyclin O (Ccno) functions during deuterosome‐mediated centriole amplification of multiciliated cells (2015)
Figure 3

Ccno is dynamically expressed from early phases of ciliogenesis and Ccno deficiency impacts on the transcriptional programme for ciliogenesis

A–H X‐Gal‐staining of (A–C) control and (D–H) CcnoTA/+ embryonic lungs at indicated stages showing onset of Ccno expression from E13 in the proximal trachea and in the main bronchi. Expression is extending to more distal regions, and from E16 staining is also found in bronchioli.
I X‐Gal‐staining indicating LacZ expression from the CcnoTA/+ allele in mTEC cultures after 5 days of differentiation‐onset by switching to air–liquid interface (ALI) conditions (ALI d5).
J Transcript levels of Ccno and indicated genes with known functions for the generation of multiple cilia during mTEC differentiation. mRNA levels from three independent experiments were measured by qRT–PCR and levels of expression set as 1 on day 0 of ALI cultures. Scales for Ccno and Deup1 are indicated on the left, and for Cep152, Cep63 and Ift57 on the right side.
K Relative mRNA expression levels for indicated genes were measured by qRT–PCR in wild‐type and Ccno‐deficient mTEC cultures at indicated days after differentiation‐onset. Relative values were calculated as in (J) relative to day 0 of ALI cultures.

Data information: Scale bars: 500 μm in (A–H) and 250 μm in (I).
Cyclin O (Ccno) functions during deuterosome‐mediated centriole amplification of multiciliated cells (2015)
Figure 4

Ccno‐deficient MCCs at embryonic stages show disturbed ciliogenesis and ultrastructural defects of deuterosome and centrioles

A–B' SEM of tracheae from (A, A') wild‐type and (B, B') CcnoRA/RA embryos at E17. In comparison to wild‐type MCCs, Ccno‐deficient MCCs almost completely lack formation of multiple cilia and only single, or doublet (arrow) cilia are observed at E17.
C–E The lack of multiple cilia at E17 stages is reflected by the almost complete absence of acetylated α‐tubulin staining as marker for axonemes in (D) Ccno‐deficient MCCs of E17 embryonic trachea as quantified in (E). FOV, field of view.
F–I TEM of MCCs from E17 embryonic tracheae shows deuterosomes with forming procentrioles in (F, F', H) Ccno‐proficient and (G, G', I) Ccno‐deficient MCCs. Asterisks in (F, G) indicate microvilli that extend from the apical cell surface. (F', G') Deuterosomes in Ccno‐deficient MCCs are significantly enlarged (identical size bars in F' and G') and show irregular morphology, which is different to the annular shape of wild‐type deuterosomes as seen in (F', H). Procentrioles (arrowheads in F' and I) that are found at deuterosomes of (I) Ccno‐deficient MCCs were recurrently found being shorter and appeared less structured in comparison to (F', H) wild‐type cells.
J The average length of procentrioles was quantified for wild‐type and Ccno‐deficient MCCs of E17 tracheae (49 procentrioles in n = 3 independent samples for wild‐types and 47 procentrioles in n = 3 independent samples for Ccno‐deficient MCCs).
K The diameter of deuterosomes (41 deuterosomes in n = 3 independent samples for wild‐types; 38 deuterosomes in n = 5 independent samples for Ccno‐deficient MCCs) was measured in wild‐type and Ccno‐deficient MCCs of E17 tracheae.

Data information: Error bars indicate the standard error of the mean (s.e.m.). P‐values were calculated by a two‐sided Student's t‐test in (E, J, K). Scale bars: 10 μm in (A, B), 4 μm in (A', B'), 10 μm in (C, D), 500 nm in (F, G) and 200 nm in (F', G'‐I).