Extended Data Fig. 8: Related to Figure 4. SP9 penetrates epithelial cells when conjugated to CPPs and inhibit mucin secretion from airway epithelium cells.
From: Inhibition of calcium-triggered secretion by hydrocarbon-stapled peptides
a, Representative confocal images (z-sections) of fixed HAE cells treated with SP9-Cy3 or SP9-Cy3 conjugated to CPPs or biotin. Biotin-SP9-Cy3 was bound to streptavidin-conjugated C2 or CRM197. The experiment was repeated twice with ALI cultures from different donors with similar results. Scale bar, 10 µm. b, The diagram illustrates the analysis of intracellular localization of MUC5AC, Cy3, and DAPI in airway secretory cells. Fluorescence intensities of DAPI, AlexaFluor 488 (MUC5AC) and Cy3 were analysed within individual MUC5AC+ cells at each z-section, normalized and fluorescence intensity traces calculated along the basolateral to apical cell axis. c, Representative western blot immunofluorescence images for MUC5AC on apical surface of untreated HAE cells (control 1 and 2) or HAE cells treated with 100 μM SP9-Cy3, PEN-SP9-Cy3, TAT-SP9-Cy3, PEN-P9-Cy3, or TAT-P9-Cy3 for 24 h before stimulation. Wash represents MUC5AC accumulated during culture and before start of experiment. Baseline represents unstimulated MUC5AC secretion during a 15 min period after removal of accumulated MUC5AC and experimental represents MUC5AC secreted within 15 min of stimulation with (ATP) or without (no ATP) 100 µM ATP. Lysate represents MUC5AC within HAE cells at the end of the experiment. Cells were treated with IL-13 to induce mucous metaplasia. All original blots are shown in Supplementary Fig 1b. d, Box plots and data points show the ratio of experimental / baseline secretion (fold increase of stimulated secretion over baseline secretion) following 24 h preincubation with 100 μM of the respective peptides. Numbers below box-plots indicate n for each condition, representing individual ALI cultures derived from 4 donors for each condition. * p = 0.046 for HAE cells treated with 100 μM PEN-SP9-Cy3, and p = 0.016 for HAE cells treated with 100 μM TAT-SP9-Cy3, assessed by two-way ANOVA followed by post-hoc Dunnett`s test.
