Fig 1: 4-PBA inhibits IL-25-induced epithelial barrier damage and suppresses apoptosis of 16HBE cells associated with PERK pathway. (a) The apoptotic cells were determined by flow cytometry using Annexin V/Propidium iodide (PI) staining. (b) The percentages of apoptotic cells were analysed. (c–f) Confocal laser immunofluorescence photomicrographs of ZO-1 and E-cadherin were measured (original magnification, ×400). Epithelial barrier function was detected by TEER measurement (g) and FITC-DX paracellular flux assay (h,i) The expression of p-PERK, PERK, p-eIF2a, BIP and CHOP were measured using immunoblotting. Relative changes in the density of BIP (j), CHOP (k) and ß-actin, p-PERK and PERK (l) were analysed. Blots in panel (g) have been cropped, and full blots are presented in Supplementary Fig. S5. Data were represented as means ± SEM (n = 6, one-way ANOVA with Tukey’s post hoc, significant differences were defined as p < 0.05).
Fig 2: IL-25 induces ERs in 16HBE cells. (a) Expression of BIP and CHOP were measured by immunofluorescent staining with BIP and CHOP antibody. DAPI was used for staining nucleus. TRICT-conjugated secondary abs (red) and FITC-conjugated secondary abs (green) were used for binding confocal laser scanning microscopy (original magnification, ×400). Fluorescence intensity of BIP (b) and CHOP (c) were quantified at 10 random fields. The mRNA levels of CHOP induced by IL25 were detected at a dose-dependent manner (d) (0 ng/ml, 1 ng/ml, 10 ng/ml, 100 ng/ml) and a time-dependent manner (e) (0 h, 4 h, 12 h, 24 h). All data were represented as means ± SEM (n = 6, one-way ANOVA with Tukey’s post hoc, significant differences were defined as p < 0.05).
Fig 3: Specific-allergen Immunotherapy increases the level of Treg cells and decreases level of IL-25. (a) CD4+ CD25+ Foxp3+ Treg cells in spleen tissue of mice were detected using flow cytometry. (b) The percentage of CD4+ CD25+ Foxp3+ Treg cells of each mice were shown in the scatter plot. 8 × 104 splenocytes were analysed in each sample. (c) The expression of IL-25 in BALF was measured using ELISA. (d) The expression of IL-25 in lung tissue was detected by western blots. (e) Relative changes in the density of IL-25 and ß-actin was analysed. Blots in panel (d) have been cropped, and full blots are presented in Supplementary Fig. S3. All data were represented as means ± SEM (n = 6, one-way ANOVA with Tukey’s post hoc, significant differences were defined as p < 0.05).
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