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Fig. 3 | Journal of Translational Medicine

Fig. 3

From: Pathogenesis of chronic rhinosinusitis with nasal polyps: role of IL-6 in airway epithelial cell dysfunction

Fig. 3

Study of the effect of ILs on the differentiation process after wound closure. a Diagram of well cutting and immunolabeling made in the three different thirds of wells (CK14 in the upper third, MUC5AC in the intermediate third and ATA in the lower third) and method of analysis: Effects of ILs on differentiation in HNEC cultures after wound closure (2 (F2), 7 (F7) and 12 (F12) days after wound closure) was characterized by immunolabeling: each well was separated in three areas for each immunolabeling marker before IL-staining in order to avoid mixed antibodies contaminations (by cutting each well in three areas): either with mouse monoclonal anti-cytokeratin 14 (CellMarque 314 M-14, 1/100) (picture a-1) as a basal cell marker, mouse monoclonal anti-mucin-5AC (Abcam ab3649, 1/200) (picture a-2) as a goblet cell marker, or mouse monoclonal anti-acetylated α-tubulin (Abcam ab24610, 1/500) (picture a-3) as a ciliated cell marker, before being revealed by a secondary goat anti-mouse Alexa Fluor- 488 antibody (Molecular Probes 1/500). Nuclei were stained by DAPI. In order to evaluate the “wound” effect and overcome the IL effect on healthy epithelium, we measured the ratio of the number of nuclei stained by DAPI or the percentage of cells positive for each immunolabeling (ATA, MUC5AC, and CK14) in two wounded areas on the number of nuclei for the DAPI in the corresponding two non-wounded control areas of the same well. be Comparison of ratios (percentage of positive cells in wounded area/Percentage of positive cells in non-wounded control areas) after 2, 7 and 12 days after wound closure between culture wells modulated by each IL (IL-5, IL-6, IL-9, IL-10) and not (controls wells). b Comparison of nuclei ratios (wounded areas/non-wounded areas) between culture wells with IL and without *p < 0.05. c Comparison of ciliated cell ratios (wounded areas/non-wounded areas) between culture wells with IL and without *p < 0.05. d Comparison of goblet cell ratios (wounded areas/non-wounded areas) between culture wells with IL and without *p < 0.05. e Comparison of basal cell ratios (wounded areas/non-wounded areas) between culture wells with IL and without *p < 0.05

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