Fig. 4: SP9 inhibits mucin secretion from airway epithelial cells.
From: Inhibition of calcium-triggered secretion by hydrocarbon-stapled peptides
a, The design of synthesized SP9 with biotin or CPPs conjugated to the N terminus and Cy3 to the C terminus, respectively. Biotin–SP9–Cy3 was bound to streptavidin-conjugated C2 or CRM197. b, Diagram of the analysis of cumulated Cy3 intensities within individual HAE cells. c, Confocal collapse (projected) images of fixed HAE cells that were treated with SP9–Cy3 or SP9–Cy3 conjugated to bacterial toxins (C2, CRM197) or CPPs. Scale bar, 10 µm. The experiment was independently repeated twice with air–liquid interface (ALI) cultures from different donors with similar results. d, Quantitative analysis of intracellular Cy3 fluorescence for each peptide in MUC5AC+ HAE cells. Box plots and data points are shown for n cells (indicated below each box plot). Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by post hoc Dunnett’s test; ***P < 0.0001. e, Schematic of the peptide application and sample collection from HAE cells maintained under ALI conditions. f, Representative western blot immunofluorescence images for MUC5AC on an apical surface of untreated HAE cells (control), HAE cells treated with 10 μM of SP9–Cy3, or 10 μM of either SP9–Cy3 or P9–Cy3 conjugated to CPPs for 30 min before stimulation. Basl., MUC5AC secretion during a 30 min period before stimulation (baseline). Exp., MUC5AC secreted within a 30 min experimental period with (IL-13 + ATP) or without (IL-13) stimulation of HAE cells with 100 µM ATP. Cells were treated with IL-13 to induce mucous metaplasia. All of the original blots are shown in Supplementary Fig. 1a. g, h, The ratio of baseline to reference wash secretion (fold increase in baseline secretion over reference secretion) (g) and the ratio of experimental to baseline (fold increase in stimulated secretion over baseline secretion) (h) for each condition in f. The numbers below the box plots indicate n for each condition, representing individual ALI cultures derived from four donors for each condition. Statistical analysis was performed using two-way ANOVA followed by post hoc Dunnett’s test; *P = 0.013.
