Journal: Nature

Article Title: Inhibition of calcium-triggered secretion by hydrocarbon-stapled peptides

doi: 10.1038/s41586-022-04543-1

Figure Lengend Snippet: 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. . 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.

Article Snippet: Primary HAE cells from several donors were obtained from Promocell at passage 2 or isolated from fresh tissues that were obtained during tumour resections or lung transplantation with fully consent of patients (Ethics approval: ethics committee Medical School Hannover, project no. 2701-2015).

Techniques: Synthesized, Fluorescence, Western Blot, Immunofluorescence, Derivative Assay

Journal: Nature

Article Title: Inhibition of calcium-triggered secretion by hydrocarbon-stapled peptides

doi: 10.1038/s41586-022-04543-1

Figure Lengend Snippet: 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 . 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.

Article Snippet: Primary HAE cells from several donors were obtained from Promocell at passage 2 or isolated from fresh tissues that were obtained during tumour resections or lung transplantation with fully consent of patients (Ethics approval: ethics committee Medical School Hannover, project no. 2701-2015).

Techniques: Fluorescence, Western Blot, Immunofluorescence, Derivative Assay