Journal: Mucosal immunology

Article Title: Airway structural cells regulate TLR5-mediated mucosal adjuvant activity.

doi: 10.1038/mi.2013.66

Figure Lengend Snippet: Figure 4 Flagellin is rapidly and totally degraded in airways conducts. C57BL/6 mice (n¼ 3–6) were treated intravenously or intranasally (i.n.) with flagellin. Spleen (a) and lung (b) were sampled at 2 h and Il22 mRNA levels were determined by quantitative PCR. Levels of mRNA in mock animal were arbitrarily set to 1 and used to calculate relative gene expression. (c–g) Mice (n ¼ 3–4) were stimulated i.n. with 1mg (d, e, g) or 10mg (c, f) flagellin and lungs and bronchoalveolar lavage (BAL) of mock or flagellin-treated mice were sampled at indicated time. " " and "þ " indicate BAL from mock animals and BAL supplemented ex vivo with flagellin, respectively. In panels c–e experiments were conducted on NMRI mice, whereas Tlr5 / and C57BL/6 (WT) animals were used for panels (f, g). (c) Kinetic of flagellin degradation. Flagellin (upper panel) or endogenous IgG (lower panel) were analyzed by SDS–polyacrylamide gel electrophoresis and immunoblot. For each time, the immunoblot for two animals were shown. (d) Concentration of flagellinwasestimated byELISA.(e)Timecourse analysisofflagellinactivity in sampled BAL. Epithelial cells Caco-2 were stimulated 24h with BAL isolated from flagellin-treated mice sampling at indicated time or medium as control ( ). Levels of IL-8 in supernatants were determined by ELISA. Resultsareexpressedasmean±s.d.Statisticalsignificance(*Po0.05)was assessed by Mann–Whitney test compared with phosphate-buffered saline (a, b) or t0 (e) group. (f) Flagellin degradation in BAL 2h after nasal administration was analyzed by flagellin-specific immunoblot. For each group, the immunoblot for 3 animals were shown. (g) Concentration of flagellin was estimated by ELISA at 2 and 6 h. ND, not done.

Article Snippet: The normal human bronchial epithelial cell BEAS-2B was grown in complete epithelial cell culture medium (Promocell GmbH, Heidelberg, Germany).

Techniques: Real-time Polymerase Chain Reaction, Gene Expression, Ex Vivo, Polyacrylamide Gel Electrophoresis, Western Blot, Concentration Assay, Isolation, Sampling, Control, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Saline

Journal: Mucosal immunology

Article Title: Airway structural cells regulate TLR5-mediated mucosal adjuvant activity.

doi: 10.1038/mi.2013.66

Figure Lengend Snippet: Figure 7 Airway epithelial cells replicate the lung signature to flagellin (a, b) Transcripts analysis of microdissected lung compartments. C57BL/6 mice (n ¼ 2–3) were treated i.n. with phosphate-buffered saline or flagellin 2 h. Bronchoalveolar lavage (BAL) and lungs were sampled. Lungs were prepared for laser microdissection of bronchial epithelium and alveolar compartment. Level of Ccl20 mRNA (a) and others proinflammatory genes transcripts (b) in bronchial epithelium and whole alveolar compartments, BAL cells or total lung were determined by quantitative PCR (qPCR). (c, d) Activation of human epithelial cells by flagellin treatment. Normal bronchial epithelial cell line BEAS-2B were stimulated 1 h with flagellin (c). Bronchial and alveolar epithelial cell line, 16HBE and A549, respectively, were stimulated 2 h with flagellin (d). Total RNA was extracted and proinflammatory gene expression analyzed by qPCR. Levels of mRNA in mock animals or cells were arbitrarily set to 1 and used to calculate relative gene expression of flagellin-treated animals or cells. Results are expressed as mean±s.d. Statistical significance (*Po0.05) was assessed by Mann–Whitney test.

Article Snippet: The normal human bronchial epithelial cell BEAS-2B was grown in complete epithelial cell culture medium (Promocell GmbH, Heidelberg, Germany).

Techniques: Saline, Laser Capture Microdissection, Real-time Polymerase Chain Reaction, Activation Assay, Gene Expression, MANN-WHITNEY

Journal: Brain : a journal of neurology

Article Title: Connexin43 mimetic peptide reduces vascular leak and retinal ganglion cell death following retinal ischaemia.

doi: 10.1093/brain/awr338

Figure Lengend Snippet: Figure 3 Three hours of hypoxia and 6 h of reperfusion lead to significant endothelial cell death in vitro in rat brain microvascular endothelial cells (RBMVEC; A). Non-specific gap junction blocker carbenoxolone, non-specific hemichannel blocker LaCl3, and connexin43 (Cx43) mimetic peptide protected endothelial cells against hypoxic injury, with the number of viable cells significantly higher than no treatment, while scrambled peptide did not have any protective effects. The number of viable cells was expressed as percentage of the control without hypoxia. Hypoxia and reperfusion also lead to significant propidium iodide dye uptake into primary rat brain microvascular endothelial cells indicating open hemichannels (B). Carbenoxolone, LaCl3 and connexin43 mimetic peptide significantly prevented dye uptake compared to no treatment, indicating hemichannel closure, while scrambled peptide did not have any effect. Stars denote statistical significance when compared to the control group or compared between groups in brackets; P 5 0.05.

Article Snippet: Rat brain microvascular endothelial cells (R840K-05a, Cell Applications) were plated into 24-well plates (1 105 cells/well) in rat brain microvascular endothelial cell growth media (R819K-500, Cell Applications) and allowed to settle for 16 h. Medium was then removed and replaced with Dulbecco’s Modified Eagle’s Medium/F12 containing 0.5% foetal bovine serum and 1% glutamine.

Techniques: In Vitro, Control

Journal: Nature cell biology

Article Title: A complex secretory program orchestrated by the inflammasome controls paracrine senescence

doi: 10.1038/ncb2784

Figure Lengend Snippet: (a) Co-culture with cells undergoing OIS induces the arrest of normal IMR90 cells. Normal IMR90 human fibroblasts expressing the fluorescent marker mCherry (IMR90 Cherry), were mixed with IMR90 ER:RAS cells. When indicated 200 nM 4OHT was added to activate ER:RAS. Growth curves represent the number of IMR90 ER:RAS (left) or IMR90 Cherry cells (right) present in the co-cultures. Data is a representative experiment of n=2 independent experiments for days 0-7 and mean +/− s.d. of n= 3 independent experiments for day 8. The source data for 2 independent experiments and statistics for day 8 (Student’s t-test) are provided in . (b) Co-culture with IMR90 MEK:ER cells induces the arrest of normal IMR90 cells. The percentage of positive BrdU cells for each population in the cocultures 4 days after 4OHT induction is shown. Data is a representative experiment. The source data for 2 independent experiments are provided in . (c) IMR90 ER:RAS or IMR90 ER cells were co-cultured with normal IMR90 Cherry fibroblasts. BrdU incorporation at day 7 shows that co-culture with IMR90 ER:RAS but not with IMR90 ER induces the arrest of normal IMR90 Cherry cells (centre). Data is a representative experiment. The source data for 2 independent experiments are provided in . Representative images are shown (right). Scale bar, 30 μm. (d) Normal HMEC or IMR90 cells suffer growth arrest when co-cultured with HMEC cells undergoing OIS. HMEC-hTERT (centre) or IMR90 (right) cells expressing Cherry as a fluorescent marker were co-cultured with HMEC-TERT ER:RAS or HMEC-TERT vector cells in the presence of 100 nM 4OHT. Growth curves showing growth arrest of HMEC-TERT Cherry (centre) or IMR90-Cherry cells (right) are shown. Data is a representative experiment.

Article Snippet: HMEC-hTert cells were cultured in mammary epithelial cell growth media (PromoCell).

Techniques: Co-Culture Assay, Expressing, Marker, Cell Culture, BrdU Incorporation Assay, Plasmid Preparation

Journal: Nature Communications

Article Title: Structure and function relationships of mucociliary clearance in human and rat airways

doi: 10.1038/s41467-025-57667-z

Figure Lengend Snippet: a Airway branching generations in human and rat investigated in this study included BG0-6 in humans and BG0-5 in rats. b Workflow for imaging luminal epithelial cell type composition and ciliary beat and clearance function in airway samples. Schematic: Created in BioRender. Nawroth, J. (2025) https://BioRender.com/a01i578 ). c Example IF staining of cilia (ATUB, magenta) and secretory cells (SCGB1A1, green; MUC5AC, gray) in human and rat airway epithelium in BG0 (trachea) and BG5/6. Scale bar: 20 µm. d Quantification of luminal cell proportions labeled with ATUB (ciliated cells) or with MUC5AC and/or SCGB1A1 (secretory cells) as a function of airway branching generation in human and rat airway epithelium. Inset: Percentage of human secretory cell population positive for only MUC5AC (gray), only for SCGB1A1 (green), or for both (white) as a function of branching generation. Solid line: mean, shaded region: SEM. Numbers of human donors (2–3 FOVs each): BG0, n = 3; BG1, n = 3; BG2, n = 5; BG3, n = 3; BG4, n = 7; BG5, n = 2; BG6, n = 2; Numbers of rat donors (2–3 FOVs each): BG0, n = 5; BG1, n = 3; BG2, n = 2; BG4, n = 1; BG5, n = 1. For full donor information see Supplementary Tables and . Source data for ( d ) are provided as a file.

Article Snippet: SAGM: Small Airway Epithelial Growth Medium (Promocell, C-21170) containing 50 nM EC23; and 5. mAir: a 1:1 mix of Dulbecco’s modified eagle medium and Airway epithelial cell growth medium (AECGM, PromoCell, C-21160) with AECGM supplements and 50 nM EC23 (previously described in ref. ).

Techniques: Imaging, Staining, Labeling

Journal: Nature Communications

Article Title: Structure and function relationships of mucociliary clearance in human and rat airways

doi: 10.1038/s41467-025-57667-z

Figure Lengend Snippet: a Representative measurement of ciliary beat frequency (CBF) and associated particle clearance trajectories and speed in a human airway epithelial sample (BG2). b Same measurements in rat airway epithelial sample (BG1). Scalebar in ( a , b ): 100 µm. c Quantification of average CBF, particle clearance speed, and clearance per beat (CPB) in human airways BG0-6 and rat airways BG0-1. Number of human donors: n = 4. Number of rat donors: n = 6. d left: Clearance directionality as a function of distance in human (red) and rat (blue) airways. Thick lines are average curves. Right: Mean directionality over a flow distance of 80 µm. Number of human donors n = 4; number of rat donors: n = 4. Boxplots: Each solid dot is the mean value of one donor (1–3 BGs, 2–4 FOVs each); red line indicates median, bottom and top edges of the box indicate 25th and 75th percentiles, whiskers indicate minimum and maximum; significance was assessed with two-sided unpaired t-test. Source data for ( c , d ) are provided as a file.

Article Snippet: SAGM: Small Airway Epithelial Growth Medium (Promocell, C-21170) containing 50 nM EC23; and 5. mAir: a 1:1 mix of Dulbecco’s modified eagle medium and Airway epithelial cell growth medium (AECGM, PromoCell, C-21160) with AECGM supplements and 50 nM EC23 (previously described in ref. ).

Techniques:

Journal: Nature Communications

Article Title: Structure and function relationships of mucociliary clearance in human and rat airways

doi: 10.1038/s41467-025-57667-z

Figure Lengend Snippet: a Representative IF-images of primary human airway epithelial cultures from 1 donor grown in different differentiation media for 28 days at ALI and stained for cilia (ATUB, magenta) and secretory cell markers (SCGB1A1, green; MUC5AC, white). Scalebar, 40 µm. b Average luminal cell type composition based on IF-staining (in vitro: N = 4–7 donors, 2 inserts each with 3–8 FOVs each; ex vivo: 9 donors, 1–3 BGs, 2–3 FOVs each). c Mapping of IF-staining data of in vitro and ex vivo samples onto three dimensions. y- axis: percentage of MUC5AC+ cells; x-axis: cilia coverage, i.e., percentage of ciliated (ATUB + ) cells; circle diameter: ratio of SCGB1A1+ to MUC5AC+ cell percentages. d Average percentage of MUC5B expressing cells and e relative percentage of MUC5B expressing cells that also express either SCGB1A1 or MUC5AC from IF-stainings (in vitro: n = 2 (SAGM, BD) to n = 4 (PC, PC-S, mAir) donors, 1–2 inserts each with 1–6 FOVs each; ex vivo: n = 3 donors, BG0, 2–4 FOVs each). Boxplot: Each dot represents mean of one donor; red line indicates median, bottom and top edges of the box indicate 25th and 75th percentiles, whiskers indicate minimum and maximum. Source data for ( b – e ) are provided as a file.

Article Snippet: SAGM: Small Airway Epithelial Growth Medium (Promocell, C-21170) containing 50 nM EC23; and 5. mAir: a 1:1 mix of Dulbecco’s modified eagle medium and Airway epithelial cell growth medium (AECGM, PromoCell, C-21160) with AECGM supplements and 50 nM EC23 (previously described in ref. ).

Techniques: Staining, In Vitro, Ex Vivo, Expressing

Journal: Nature Communications

Article Title: Structure and function relationships of mucociliary clearance in human and rat airways

doi: 10.1038/s41467-025-57667-z

Figure Lengend Snippet: a Quantitative analysis of particle CPB and directionality in primary human airway epithelial cultures grown in different differentiation media for 28 days at ALI compared to human and rat benchmark data. Number of donors (2–3 inserts each with 6 FOVs each) for BD, mAir, SAGM, PC, PC-S, respectively: n = 5, 4, 4, 5, 4. b Quantitative analysis of ciliary beat metrics in airway cells cultured and visualized as in ( a ). Number of donors (2–3 inserts each with 6 FOVs each) for BD, mAir, SAGM, PC, PC-S, respectively: Cilia Coverage, n = 7, 4, 4, 7, 4; Ciliary Beat OP, n = 4, 4, 2 (1 not measurable), 4, 4; Ciliary Beat Amplitude: n = 4, 4, 3, 4, 4; Cilia Length, n = 4, 4, 4, 4, 4; Ciliation Gap, n = 4, 4, 3, 4, 4; Crystalline OP, n = 4, 4, 3, 4, 4. Boxplots in ( a , b ): Each solid dot is the mean value of one donor (1–3 BGs, 2–4 FOVs each); red line indicates median, bottom and top edges of the box indicate 25th and 75th percentiles, whiskers indicate minimum and maximum. Dotted lines indicate average human and rat benchmark values. c Top: Predicted CPB and clearance directionality in in vitro cultures compared to predicted human airway performance (red). Shaded regions indicate uncertainty based on spread of input metrics. Measured mean values per donor are overlaid (dots). Bottom: Predicted change in CPB and directionality of in vitro cultures in different media if an individual cilia input parameter is set to match ex vivo values. Source data for ( a – c ) are provided as a file.

Article Snippet: SAGM: Small Airway Epithelial Growth Medium (Promocell, C-21170) containing 50 nM EC23; and 5. mAir: a 1:1 mix of Dulbecco’s modified eagle medium and Airway epithelial cell growth medium (AECGM, PromoCell, C-21160) with AECGM supplements and 50 nM EC23 (previously described in ref. ).

Techniques: Cell Culture, In Vitro, Ex Vivo