Figure 7.

IL-13 alone promoted phosphorylation of STAT6 and maturation and proliferation of SPEM cells. (A) Representative bright field images of Matrigel domes containing Meta1 gastroids treated with either rIL-13 or vehicle solution (BSA). Enlarged images from H&E staining show cellular distribution and height differences between the rIL–13-treated and the BSA-treated Meta1 gastroids. Quantitation of matched gastroid diameters showed significantly higher sizes in Meta1 gastroids treated with rIL-13 compared with control after 2 days of treatment. Scale bar: 1000 μm for brightfield and 100 μm for H&E. Mean ± SD (N = 3 independent experiments). ∗∗P < .01, ∗∗∗∗P < .0001. (B) Immunoblotting shows significant up-regulation of phosphorylated STAT6 in Meta1 gastroids treated with different concentrations of rIL-13 when compared with BSA. Quantitation showed rIL-13–dose-dependent increase. Mean ± SD (N = 3 independent experiments). ∗P < .05. (C) Immunofluorescence staining of Meta1 gastroids cross sections for SPEM-related markers, AQP5 and TFF2. Quantitation of AQP5 and TFF2 double positive cells demonstrated significant increase of proportion of mature SPEM cells in rIL-13–treated Meta1 gastroids. Yellow arrowheads point at double positive mature SPEM cells. Scale bar: 100 μm. Mean ± SD (N = 3 independent experiments). ∗∗∗P < .001. (D) Immunofluorescence staining of Meta1 gastroids cross sections for a proliferation marker, Ki67, and SPEM-related markers, MUC6 and CD44v9. Quantitation of MUC6 and CD44v9 double positive cells demonstrated significant increase of proportion of mature SPEM cells in rIL-13–treated Meta1 gastroids. Moreover, quantitation of MUC6, CD44v9, and Ki67 triple positive cells demonstrated significant increase of proportion of proliferative SPEM cells. Yellow arrowheads point at triple positive mature and proliferative SPEM cells. Scale bar: 100 μm. Mean ± SD (N = 3 independent experiments). ∗∗P < .01, ∗∗∗P < .001.