Figure 4.

The GNAS mutant shows tropism for ductal-lineage epithelia

(A) Immunoblot analysis for GNAS expression and phosphorylation of GNAS downstream effectors in DUs and ACs (n = 3, independent cultures).

(B) Phase-contrast images of DUs and ACs expressing wild-type (WT) GNAS and GNAS^R201C. (-)DOX, without doxycline treatment; (+)DOX, with doxycycline treatment. Scale bar, 100 μm.

(C) Change in size, measured as the total area of organoids expressing GNAS^WT or GNAS^R201C. Box-and-whisker plot, range 5%–95%; central lines, median values (n > 100, combination of three independent cultures).

(D) Percentage of organoids surpassing the 99^th percentile of control organoid (no transgene expression) size (n = 3 independent cultures).

(E) Organoid cell number changes induced by GNAS^WT and GNAS^R201C as detected by CellTiter. Bar charts represent the mean and standard deviation of measurements from three independent cultures; gray dots represent individual measurements.

(F) Immunofluorescent staining of polarity proteins in organoids without (−DOX) and with (+DOX) GNAS^R201C expression. ZO1, red; collagen IV, green; DAPI, blue. Scale bar, 100 μm.

(G) Expression of SOX9 in organoids without (−DOX) and with (+DOX) GNAS^R201C expression. SOX9, red; DAPI, blue.

(H) AB (pH 2.5) staining of organoids without (−DOX) and with (+DOX) GNAS^R201C expression. Scale bar, 100 μm.

(I) Expression of E-cadherin (green) and MUC2 (red) in DUs without (−DOX) and with (+DOX) GNAS^R201C expression. Scale bar, 50 μm.

(J) Percentages of DUs expressing MUC2 (top chart) or MUC5AC (bottom chart). Bar charts represent the mean and standard deviation of measurements from three independent cultures; gray dots represent individual measurements.

(K and L) RNA expression of lineage markers in DUs (K) and ACs (L) altered by GNAS^R01C expression. Floating column charts represent RNA measurements from quantitative PCR (n = 3 technical repeats), hinges represent maximal and minimal values, central lines indicate mean values, and dots represent individual measurements. ^∗∗∗p < 0.001; ^∗∗, 0.001 < p < 0.01; ^∗, 0. 01 < p < 0.05; N.S., p > 0.05.