FIGURE 4.

Inhibiting PORCN results in a reduced EV secretion in human bronchiolar organoids. (A–C) Immunostaining of human bronchiolar organoids for the indicated molecules (confocal microscopic images). For panels (B,C), quantification of the images is shown for different patient-derived organoids (^#, see Supplementary Information). (D) Immunostaining of β-catenin (confocal microscopic image). Arrows indicate the nuclear localization of β-catenin. (E) Expression profile of selected Wnt genes in the organoids and fibroblasts (RT-qPCR, matched samples were used). Results normalized to HPRT1 housekeeping were z-score transformed. Red and green colors indicate higher and lower RNA levels, respectively. (F) Relative RNA level of AXIN2, LGR6, and TROY after treating organoids with LGK974 (RT-qPCR, n = 3). Data were normalized to HPRT1 housekeeping and then compared to untreated samples (Ctr). (G) The percentage of KI67+ cells in the organoids with/without treatment with the PORCN inhibitor (confocal microscopic images were quantified). (H) EV number change in the presence of LGK974 (NTA, n = 4). Data normalized to 10⁶ cells were compared to the untreated control (Ctr). (I) Fold change in the percentage of anti-CD63 and anti-CD81-coated beads incubated in the conditioned medium derived from untreated (Ctr) or treated (LGK) organoids and detected with anti-CD63 or anti-CD81 antibody, respectively. Note that medium control (without EVs) was always <1%. All data were normalized to 10⁶ cells before comparisons (flow cytometry, n = 5). One-sample t-test after log10 transformation of fold change data (F,H,I) or Mann–Whitney test for within-sample comparisons (G) was used with *p < 0.05 and ***p < 0.005. Scale bars: 50 μm (A–C) or 20 μm (D).