Journal: PLOS Pathogens

Article Title: CD56-mediated activation of human natural killer cells is triggered by Aspergillus fumigatus galactosaminogalactan

doi: 10.1371/journal.ppat.1012315

Figure Lengend Snippet: CD56 expression on naïve NK cells (Control) and NK cells stimulated for 3 h with A . fumigatus germ tubes, depending on pre-treatment of the germ tubes with proteinase K (A) or periodate (B). Data for 3 independent donors are shown. (C) Significantly differentially expressed genes after 3-h stimulation of NK cells with A . fumigatus germ tubes pre-treated (60 min) with periodate or untreated (0 min). Abbreviations: adj. p = adjusted p-value, D = donor, fc = fold change. (D) Volcano plot summarizing transcriptional changes in NK cells stimulated with periodate-treated A . fumigatus germ tubes compared to cells stimulated with untreated germ tubes. Genes with a log2 fold change > 0.5 and an adjusted p-value < 0.05 are highlighted. Selected immune-related genes have been labelled. (E) CD56 expression on naïve NK cells (Control) and NK cells stimulated for 3 h with A . fumigatus germ tubes, depending on the duration of periodate pre-treatment of the germ tubes. Representative histograms and data for cells from 5 independent donors are shown. (F) Chemokine and perforin release by naïve NK cells (Control) and NK cells stimulated for 3 h with A . fumigatus germ tubes, depending on the duration of periodate pre-treatment of the germ tubes. (A-B; E-F) Columns and error bars indicate means and standard deviations, respectively. Repeated measures one-way analysis of variance with Tukey’s post-hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Hyphae were incubated with soluble CD56 (5 μg/mL, R&D Systems, Minneapolis, MN, USA, Cat#2408-NC) or BSA (control, 5 μg/mL, Roth, Karlsruhe, Germany, #0163.2) in colorless RPMI for 2 h at 37°C, 5% CO 2 .

Techniques: Expressing, Control

Journal: PLOS Pathogens

Article Title: CD56-mediated activation of human natural killer cells is triggered by Aspergillus fumigatus galactosaminogalactan

doi: 10.1371/journal.ppat.1012315

Figure Lengend Snippet: (A) CD56 binding to fungal carbohydrates and proteins, as determined by enzyme-linked immunosorbent assay. GAG = galactosaminogalactan, RodAp = surface rodlet protein/hydrophobin. N = 3 technical replicates. One-way analysis of variance (ANOVA) with Dunnett’s post-hoc test versus Control, i.e., no coating. (B) Pull-down assay of CD56 with urea-insoluble galactosaminogalactan (PGG) or β-1,3-glucan. Lane M: Protein marker; Lane A: loading buffer extract of GAG + CD56 pellet; Lane B: supernatant of GAG + CD56; Lane C: CD56 alone; Lane D: supernatant of β-1,3-glucan + CD56; Lane E: loading buffer extract of β-1,3-glucan + CD56 pellet. (C) CD56 and CD69 expression on naïve NK cells (Control) and NK cells stimulated for 24 h with different concentrations of PGG. Representative histograms and data for cells isolated from 3 independent donors are shown. (D) Chemokine release by naïve NK cells (Control) and NK cells stimulated for 24 h with different concentrations of PGG. N = 3 independent donors. (C-D) Repeated measures (RM) one-way ANOVA with Tukey’s post-hoc test. (E) CD56, CD69, and CD107a expression on naïve NK cells (Control) and NK cells stimulated for 6 h with A . fumigatus (ATCC46645, AF) or A . nidulans (ATCC11267, AN) at different multiplicities of infection (MOIs). Representative histograms for one donor at MOI 4 are shown. (F) MOI-dependent stimulation of NK-cellular chemokine secretion after 6-h stimulation with AF or AN. (E-F) N = 3 independent donors. RM one-way ANOVA with Dunnett’s post-hoc test versus Control, i.e., unstimulated NK cells (asterisks). In addition, AF and AN stimulation at each MOI was compared using paired t-Test (hash signs). (A-F) Columns and error bars indicate means and standard deviations, respectively. */# p < 0.05, **/## p < 0.01, *** p < 0.001.

Article Snippet: Hyphae were incubated with soluble CD56 (5 μg/mL, R&D Systems, Minneapolis, MN, USA, Cat#2408-NC) or BSA (control, 5 μg/mL, Roth, Karlsruhe, Germany, #0163.2) in colorless RPMI for 2 h at 37°C, 5% CO 2 .

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Control, Pull Down Assay, Marker, Expressing, Isolation, Infection

Journal: PLOS Pathogens

Article Title: CD56-mediated activation of human natural killer cells is triggered by Aspergillus fumigatus galactosaminogalactan

doi: 10.1371/journal.ppat.1012315

Figure Lengend Snippet: (A) Representative fluorescent micrographs (z-projection of 3–4 slices with 1 μm distance, representative dataset from ≥5 independent experiments) of hyphae of wild-type (WT) A . fumigatus Af293 and two galactosaminogalactan (GAG)-deficient A . fumigatus mutants (Δ uge3 and Δ agd3 ) co-cultured with soluble CD56, followed by staining with fluorescent anti-CD56 antibody. The shape of the hyphae is indicated by dotted lines The insets represent a control image (bottom; BSA stained with anti-CD56 antibody) of the respective strain. Scale: 10 μm. (B) CD56, CD69, and CD107a expression on naïve NK cells (Control) and NK cells stimulated for 6 h with WT Af293 or the GAG-deficient A . fumigatus mutants (Δ uge3 and Δ agd3 ) at different multiplicities of infection (MOIs). Representative histograms for one donor at MOI 4 are shown. (C) MOI-dependent induction of NK-cellular secretion of granzyme B, perforin, and chemokines after 6-h stimulation with WT Af293, Δ uge3 , and Δ agd3 . (B-C) N = 3 independent donors. Repeated measures (RM) one-way analysis of variance (ANOVA) with Dunnett’s post-hoc test versus Control, i.e., unstimulated NK cells (asterisks). In addition, results for stimulation with the 3 strains at each MOI was compared using RM one-way ANOVA with Dunnett’s post-hoc test versus WT (hash signs). Columns and error bars indicate means and standard deviations, respectively. */# p < 0.05, **/## p < 0.01, ***/### p < 0.001. (D) CLSM micrographs of NK cells co-cultured with WT Af293 and Δ agd3 A . fumigatus hyphae. CD56 was stained with anti-CD56 Alexa Fluor 647 (red) to assess the CD56 localization. Germ tubes could be detected via their auto-fluorescence (cyan). Scale: 10 μm.

Article Snippet: Hyphae were incubated with soluble CD56 (5 μg/mL, R&D Systems, Minneapolis, MN, USA, Cat#2408-NC) or BSA (control, 5 μg/mL, Roth, Karlsruhe, Germany, #0163.2) in colorless RPMI for 2 h at 37°C, 5% CO 2 .

Techniques: Cell Culture, Staining, Control, Expressing, Infection, Fluorescence

Journal: PLOS Pathogens

Article Title: CD56-mediated activation of human natural killer cells is triggered by Aspergillus fumigatus galactosaminogalactan

doi: 10.1371/journal.ppat.1012315

Figure Lengend Snippet: (A) CD56 binding to fully acetylated (aPGG), fully deacetylated (dePGG), and native galactosaminogalactan (PGG), was determined by enzyme-linked immunosorbent assay (ELISA). Additional conditions with incomplete ELISA setup were included to preclude unspecific binding of the secondary antibody or anti-CD56 to the carbohydrates. N = 3 technical replicates. One-way analysis of variance (ANOVA) with Tukey’s post-hoc test was performed for each assay setup. (B) CD56, CD69, and CD107a expression on naïve NK cells (Control) and NK cells stimulated for 24 h with PGG, aPGG, or dePGG. Representative histograms for cells isolated from one donor are shown. (C) NK-cellular chemokine secretion after 24-h stimulation with PGG, aPGG, or dePGG. (B-C) N = 6 independent donors. Repeated measures (RM) one-way ANOVA with Tukey’s post-hoc test. (D) CD56, CD69, and CD107a expression on naïve NK cells (Control) and NK cells stimulated for 24 h with GalN oligomers, GalNAc oligomers, or chitosan. NK-cellular chemokine secretion after 24-h stimulation with GalN oligomers, GalNAc oligomers, or chitosan. N = 4 independent donors. RM one-way ANOVA with Tukey’s post-hoc test. (A-D) * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Hyphae were incubated with soluble CD56 (5 μg/mL, R&D Systems, Minneapolis, MN, USA, Cat#2408-NC) or BSA (control, 5 μg/mL, Roth, Karlsruhe, Germany, #0163.2) in colorless RPMI for 2 h at 37°C, 5% CO 2 .

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Expressing, Control, Isolation

Journal: PLOS Pathogens

Article Title: CD56-mediated activation of human natural killer cells is triggered by Aspergillus fumigatus galactosaminogalactan

doi: 10.1371/journal.ppat.1012315

Figure Lengend Snippet: (A) CD56 and CD69 expression of unstimulated NK cells (Control) and A . fumigatus -stimulated NK cells (6 h) depending on the fungal strain (wild type [WT] Af293 or isogenic mutants with defective GAG biosynthesis) and its enzymatic pre-treatment. (B) Secretion of granzyme B and perforin by unstimulated NK cells (Control) and A . fumigatus -stimulated NK cells depending (6 h) on the fungal strain and its enzymatic pre-treatment. (A-B) N = 6 independent donors. Repeated measures (RM) one-way analysis of variance (ANOVA) with Dunnett’s post-hoc test versus Control, i.e., unstimulated NK cells (asterisks). Additionally, enzymatic pre-treatments of A . fumigatus WT and the Δ agd3 mutant, respectively, were compared using RM one-way ANOVA with Dunnett’s post-hoc test versus no enzymatic pre-treatment (hash signs). Columns and error bars indicate means and standard deviations, respectively. */# p < 0.05, **/## p < 0.01, ***/### p < 0.001.

Article Snippet: Hyphae were incubated with soluble CD56 (5 μg/mL, R&D Systems, Minneapolis, MN, USA, Cat#2408-NC) or BSA (control, 5 μg/mL, Roth, Karlsruhe, Germany, #0163.2) in colorless RPMI for 2 h at 37°C, 5% CO 2 .

Techniques: Expressing, Control, Mutagenesis

Journal: Cell death & disease

Article Title: Cyclic increase in the ADAMTS1-L1CAM-EGFR axis promotes the EMT and cervical lymph node metastasis of oral squamous cell carcinoma.

doi: 10.1038/s41419-024-06452-9

Figure Lengend Snippet: Fig. 3 L1CAM is critical in ADAMTS1-modulated invasive ability of oral squamous cell carcinoma (SCC; OSCC) cells and was correlated with poor prognoses in patients with head and neck SCC (HNSCC). A, B HSC-3 or HSC-3M cells expressed ADAMTS1-flag, shADAMTS1, or their respective control as indicated. Cell lysates and conditioned media were collected to detect endogenous ADAMTS1 or L1CAM protein levels (A) and secreted soluble L1CAM (B), respectively using western blotting and Dot-blotting assays. C A L1CAM shRNA was transfected into ADAMTS1-overexpressing HSC-3 cells as indicated and subjected to Matrigel-invasion assays. D HSC-3 cells were treated with indicated concentrations of rhL1CAM for 24 h and subjected to Matrigel-invasion assays. C, D Multiples of differences are presented as the mean ± SD of three independent experiments. ***p < 0.001, compared to the control group; ###p < 0.001, compared to the ADAMTS1-overexpressing only group. E L1CAM expression was analyzed in 43 matched HNSCC tissues and their corresponding normal tissues using data from TCGA. F Kaplan–Meier analysis of overall survival (OS) and disease-specific survival (DSS) rates in patients with HNSCC presenting with high or low L1CAM expression using data from TCGA. G Correlation analysis of TCGA HNSCC databases (TCGA, PanCancer Atlas) using cBioPortal which revealed a positive correlation between expressions of ADAMTS1 and L1CAM. H Survival heat map showing the prognostic impacts of ADAMTS1 and L1CAM on 33 different cancer types according to the GEPIA2 database. HR hazard ratio. I Combined high ADAMTS1 expression and high L1CAM expression were correlated with the worst OS and DSS in patients with HNSCC compared to patients with other expression statuses of ADAMTS1 and L1CAM.

Article Snippet: A recombinant human L1CAM (777-NC) was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Control, Western Blot, shRNA, Transfection, Expressing

Journal: Cell death & disease

Article Title: Cyclic increase in the ADAMTS1-L1CAM-EGFR axis promotes the EMT and cervical lymph node metastasis of oral squamous cell carcinoma.

doi: 10.1038/s41419-024-06452-9

Figure Lengend Snippet: Fig. 6 Targeting ADAMTS1 by apigenin (API) resulted in suppression of the invasion of oral squamous cell carcinoma (OSCC) cells. A HSC-3, HSC-3M, and SAS cells were treated with API at 40 μM for different durations, and ADAMTS1, L1CAM, and EGFR expressions were evaluated by Western blotting. B HSC-3M cells were treated with API or infected with ADAMTS1 shRNAs for 24 h, and cell-invasive abilities were measured by a Matrigel-invasion assay. C, D HSC-3 cells were transiently transfected with a vector control or ADAMTS1-flag followed by API or vehicle treatment for an additional 24 h. ADAMTS1 expression and invasive ability in cells were respectively detected by Western blotting (C) and Matrigel-invasion assays (D). B, D Representative photographs of invaded cells (left panel) and quantification of those cells (right panel). Data are presented as the mean ± SD of three independent experiments, *p < 0.05, ***p < 0.001 vs. control cells and ##p < 0.01, vs. ADAMTS1-overexpressing only cells.

Article Snippet: A recombinant human L1CAM (777-NC) was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Western Blot, Infection, Invasion Assay, Transfection, Plasmid Preparation, Control, Expressing

Journal: Cell death & disease

Article Title: Cyclic increase in the ADAMTS1-L1CAM-EGFR axis promotes the EMT and cervical lymph node metastasis of oral squamous cell carcinoma.

doi: 10.1038/s41419-024-06452-9

Figure Lengend Snippet: Fig. 8 Schematic presentation depicting the ADAMTS1-L1CAM-EGFR axis in promoting the epithelial-mesenchymal transition (EMT) and metastasis of oral squamous cell carcinoma (OSCC). EGFR activation might be triggered by formation of the ADAMTS1-L1CAM-EGFR complex or through ADAMTS1-mediated TGF-β upregulation to subsequently induce L1CAM upregulation and L1CAM-integrin binding, resulting in induction of IL-1β secretion. Bold dashed ovals indicate hypothetical molecules that participate in the ADAMTS1-L1CAM axis to transactivate EGFR signaling, and EGFR-activated signaling may exert positive feedback regulation on ADAMTS1 expression. Cyclic increases in ADAMTS1 and EGFR activation lead to exacerbation of the EMT and invasive abilities of OSCC cells.

Article Snippet: A recombinant human L1CAM (777-NC) was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Activation Assay, Binding Assay, Expressing