Journal: medRxiv

Article Title: Charcot-Leyden Crystals drive pathology and inflammation in muco-obstructive lung disease

doi: 10.1101/2025.11.26.25341000

Figure Lengend Snippet: a , Immunostaining for neutrophils (MPO) in a mucosal biopsy taken from an internal control segment (left) and a plugged segment with CLCs present (right). b, Comparison of samples with CLCs absent or present in the cont ext of asthma and CF for IP-10 c, Immunostaining for Neutrophil Extracellular Traps (NETs) in mucus plugs without (left) and with (right) CLCs. d, Comparison of samples with CLCs absent or present in the context of asthma and CF for NETosis via ELISA (median 0.17 vs 0.19, p < 0. 0001). Points are colored by disease (asthma, sky blue; CF, vermilion) and shaped by sample type (spontaneous sputum, ●; plug, ×). Black crossbars indicate medians. Mann-Whitney U statistical test. e, Release of NETs by human peripheral neutrophils left unstimulated (PBS) or stimulated with 100µg/ml CLCs for 4 hours followed by staining of extracellular DNA with SYTOX Green Scale bar f, Detection of NETs produced by human neutrophils stimulated with 100µg/ml CLCs for 4 hours followed by immuno-staining of Citrullinated H3 (Cit-H3), Myeloperoxidase (MPO), DAPI and detection of FITC-CLCs Scale bar, 50µm g, Multiple neutrophils and NETs decorate a CLC following incubation of human neutrophils with 100µg/ml CLCs for 4 hours, shown with scanning electron microscopy scale bar h, Left panel demonstrates an example of production of extracellular DNA (DRAQ7) quantified by live cell imaging following incub ation of human peripheral neutrophils with medium (mock), PMA (100nm) or 100µg/ml CLCs for 4 hours. Quantification of percent age of DRAQ7 cells at 4 hours, quantifed from n=3 –9 positions from 2 separate experiments. i, Coating of FITC-CLCs with MPO produced by human peripheral neutrophils following co –incubation with 100µg/ml CLCs for 4 hours. Scale bar j, Interaction of dsDNA with Galectin-10 as demonstrated by Biolayer Interferometry (BLI) measurement k, Left panel shows an example experiment quantif ying the increase in elastic modulus when human neutrophils are incubat ed with 100µg/ml CLCs in the presence of a hyaluronic acid hydrogel on a cone –and-plate rheometer. Right panel shows the quantification of elastic modulus (G’) at 1Hz at 1 hour (n=2-5 from 4 separate experiments). l, An increase in the elastic (G’) modulus of a hyaluoronic acid based hydrogel following incubation with BAL supernatant retrieved from mice 24 hours post-intratracheal administration of PBS, 100µg/mouse CLCs, or 100µg/mouse CLCs + DNAse (n=2-4 mice from 2 experiments) m, Immuno-staining of neutrophils with myeloperoxidase (MPO) and Galectin-10 crystals in human mucus plug demonstrating the close interaction of neutrophils with CLCs. Scale bar = 2µm. n, Scanning electron microscopy of human neutrophils stimulated with Galectin-10 crystals for 4 hours, demonstrating the penetrance of CLCs by neutrophils and DNA. o, Scanning electron microscopy of CLCs incubated with herring sperm DNA (hsDNA) for 4 hours, demonstrating porous holes in crystals p, Scanning electron microscopy and q , t ransmission electron microscopy of CLCs in a human mucus plug where porous holes in CLCs can be seen.

Article Snippet: Interestingly, we have observed DNA binding to Galectin-10 carrying a point mutation in its carbohydrate binding site that abbrogates carbohydrate binding.

Techniques: Immunostaining, Control, Comparison, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Staining, Produced, Incubation, Electron Microscopy, Live Cell Imaging

Journal: Nucleic acids research

Article Title: The histone demethylase Kdm3a is essential to progression through differentiation.

doi: 10.1093/nar/gks399

Figure Lengend Snippet: Figure 3. Kdm3a positively regulates the expression of Dab2, FoxQ1 and Pdlim4 and this correlates with their H3K9me2 demethylation. (A) Heatmap depicting microarray expression changes (log2) of selected differentiation-associated genes after Kdm3a-knockdown in F9 cells after RA and RA+dbcAMP treatment. Differential gene expression was calculated between RNAi control F9 cells and RNAi Kdm3a cells for each time-point separately (no treatment, RA and RA+dbcAMP). Genes were ordered from the most down-regulated to the most up-regulated according to their expression changes upon Kdm3a-knockdown at 6 days after RA+dbcAMP treatment. Double appearances indicate multiple microarray probes for the respective gene (probe ID in brackets). (B) Kdm3a positively regulates the expression of Dab2, FoxQ1 and Pdlim4. Expression was analysed in RNAi control and RNAi Kdm3a F9 cells after no treatment, treatment for 3 days with RA (RA), or treatment for 3 days with RA and then for 3 additional days with RA and dbcAMP (RA+dbcAMP). RNA extracted from these cells was subjected to qRT-PCR analysis of Dab2, FoxQ1, Pdlim4 and control genes (Tcl1, Ccnd1, Serpine2, Defa1, Defcr20 and Defcr6) with gene-specific primers. Hprt was used as an internal control. All transcript levels were normalized to the level recorded for RA-treated RNAi control cells. Values represent means ± SEM (n = 3). (C) Analysis of H3K9me3/me2 modifications at the promoters of the Dab2, FoxQ1 and Pdlim4 genes after RA+dbcAMP treatment of the cells. ChIP-qPCR results from (RA+dbcAMP)-treated RNAi control cells are indicated by filled bars, those from RNAi Kdm3a F9 cells by open bars. The gene promoters analysed and the antibody used for ChIP are indicated. The normalization method used to represent the ChIP-qPCR data is fold enrichment relative to the ‘no antibody’ signal (cf. ‘Materials and Methods’ section). Data are represented as means ± SEM (n = 3).

Article Snippet: Dab2 rescue experiments Dab2 cDNA was subcloned from a commercially available clone (Origene, MC200502) into pcDNA3.1-HA (CMV promoter).

Techniques: Expressing, Microarray, Knockdown, Gene Expression, Control, Quantitative RT-PCR, ChIP-qPCR

Journal: Nucleic acids research

Article Title: The histone demethylase Kdm3a is essential to progression through differentiation.

doi: 10.1093/nar/gks399

Figure Lengend Snippet: Figure 4. Dab2 is a downstream effector of Kdm3a and contributes to progression through F9-cell terminal differentiation. F9 cells were infected with the empty pRS vector (RNAi control) or the Dab2-targeting pRS vector (RNAi Dab2). After selection, RNAi control and RNAi Dab2 F9 cells were left untreated (no treatment) or cultured with RA for 3 days (RA) or with RA for 3 days and then for 3 additional days with RA and dbcAMP (RA+dbcAMP). (A) Specific downregulation of Dab2 expression in RNAi Dab2 cells. mRNA was subjected to qRT-PCR analysis with Dab2 (left panel) or Kdm3a (right panel) specific primers. All transcript levels were normalized to Hprt and then subtracted from the level recorded for RA-treated RNAi control cells. Data are presented as means ± SEM (n = 5). (B) Dab2 is required for PE differentiation of F9 cells. RNAi Dab2 and RNAi control cells show similar morphology after RA treatment. But, RNAi Dab2 cells fail to complete terminal differentiation into PE cells upon RA+dbcAMP treatment. Cells were photographed under a phase contrast microscope. Bar = 200 mm. (C) RNAi Dab2 F9 cells still express TROMA-1 after RA+dbcAMP treatment. Immunofluorescence detection of TROMA-1 was performed on RNAi control and RNAi Dab2 F9 cells cultured on glass cover slips and treated for 6 days with RA+dbcAMP. Cytoplasmic TROMA-1 staining is absent, as expected, in RNAi control cells fully differentiated into PE cells, but still present in RNAi Dab2 cells having failed to complete PE differentiation and still at the PrE differentiation stage. Cell DNA content was visualized by DAPI staining. Bar = 40 mm. (D) Downregulation of expression of the PE differentiation marker Thrombomodulin (TM) in RNAi Dab2 cells compared to RNAi control cells. Cells were either not treated (no treatment), treated for 3 days with RA (RA), or treated for 3 days with RA and then for 3 additional days with RA and dbcAMP (RA+dbcAMP). Their mRNA was analysed by qRT-PCR. Hprt was used as an internal control. All transcript levels were corrected by the normalized level recorded for (RA+dbcAMP)-treated RNAi control cells. Values represent means ± SEM (n = 5).

Article Snippet: Dab2 rescue experiments Dab2 cDNA was subcloned from a commercially available clone (Origene, MC200502) into pcDNA3.1-HA (CMV promoter).

Techniques: Infection, Plasmid Preparation, Control, Selection, Cell Culture, Expressing, Quantitative RT-PCR, Microscopy, Staining, Marker

Journal: Nucleic acids research

Article Title: The histone demethylase Kdm3a is essential to progression through differentiation.

doi: 10.1093/nar/gks399

Figure Lengend Snippet: Figure 5. Dab2 over-expression alone is insufficient to restore a normal phenotype in RNAi Kdm3a cells. (A) Schematic representation of the transfection schedule applied during the F9-cell differentiation process. RNAi Kdm3a cells were either untransfected (timeline #1) or transfected with a Dab2 expression plasmid or a control vector at different times during the differentiation process: after 2 days of RA treatment (#2), at the start of dbcAMP treatment after 3 days of RA treatment(#4), after 4 days of treatment (#5; i.e. after 3 days of RA treatment and 1 day of

Article Snippet: Dab2 rescue experiments Dab2 cDNA was subcloned from a commercially available clone (Origene, MC200502) into pcDNA3.1-HA (CMV promoter).

Techniques: Over Expression, Transfection, Cell Differentiation, Expressing, Plasmid Preparation, Control

Journal: Frontiers in Immunology

Article Title: Secretion of the Phosphorylated Form of S100A9 from Neutrophils Is Essential for the Proinflammatory Functions of Extracellular S100A8/A9

doi: 10.3389/fimmu.2018.00447

Figure Lengend Snippet: S100A8/A9 are secreted by NETosis from both differentiated HL-60 (dHL-60) cells and purified neutrophils. (A,B) Immunofluorescent staining of neutrophil extracellular traps (NETs) from dHL-60 cells (A) or neutrophils (B) . dHL-60 cells or neutrophils were stimulated with 100 nM fMLF (middle panels) or 100 nM phorbol 12-myristoyl 13-acetate (PMA) (lower panels) for 4 h and stained for DNA (DAPI; blue staining), myeloperoxidase (MPO; green staining) or neutrophil elastase (HNE; orange staining). Pictures are representative of at least three independent experiments. (C,D) Quantification of NET release from dHL60 cells (C) or neutrophils (D) . Extracellular NET-DNA was quantified with the cell-impermeable fluorescent dye Sytox blue and NET-release was detected via fluorescence emission. NET formation was normalized to the non-stimulated control and expressed as fold induction. Results are presented as mean ± SEM of four independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: After indicated treatments, the cell impermeable DNA binding Dye Sytox blue (ThermoFischer, Ghent, Belgium) was added to the wells at a concentration of 0.5 μM to detect extracellular DNA.

Techniques: Purification, Staining, Fluorescence, Control