Figure 1.

Similar responses of WT nasospheroids using two conditions of exposure to Matrigel and differences between incubation with FSK or DMSO. (a) Nasospheroids from seven WT patients were grown in two conditions: the standard protocol or condition 1 (C1) (spheres formed in suspension and embedded in Matrigel the day before live-cell imaging) and condition 2 (C2) (spheres in Matrigel for 7 days minimum) (see details in Methods). The starting size of each nasospheroid was set at 1. At time point 0, nasopheroids were in basal conditions. For CFTR activation (FSK), 10 µM forskolin plus IBMX and amiloride (for epithelial sodium channel “ENaC” inhibition) were incubated (t = 10; black arrow). For non-stimulated spheres, dimethyl sulfoxide (DMSO) was administered. Size reduction in respect to basal size was calculated along time points up to 60 min. No significant differences were found between conditions 1 and 2 (“FSK” incubation or non-stimulated “DMSO”) (*p = 0.1563; paired t-test). Dots and triangles represent the mean fractional reduction (FR) at each time point. Bars represent standard deviation error (SEM). (b) Results from spheres grown under conditions 1 and 2 were combined. Differences between stimulus DMSO and FSK were observed from t = 10 and increased over time (t-test by GraphPad Prism and by our newly developed logarithmic-transformed measures’ model) (t = 10 min p = 0.008, t = 35 min p = 0.0.0004, t = 70 min p = 0.0002). We also observed a reduction peak when DMSO or FSK were added at time 10 (black arrow). This change could be due to a reaction of nasospheroids to the addition of compounds. Bars represent standard deviation error (SEM). Graphic created with GraphPad Prism version 6³³.