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. Author manuscript; available in PMC: 2019 Dec 28.
Published in final edited form as: Cell Rep. 2019 Sep 24;28(13):3338–3352.e6. doi: 10.1016/j.celrep.2019.08.063

Figure 4. Wnt/β-Catenin Signaling Inhibits Differentiation and Promotes Stemness in Human BCs.

Figure 4.

(A–F) Human immortalized BC (BCIs) kept in air-liquid interface (ALI) culture for up to 4 weeks. Human recombinant R-spondin2 (RSPO2) was used to activate Wnt/β-catenin signaling starting at ALI day 7 (D7). n = 3 cultures per time point and treatment.

(A) Confocal imaging of specimens stained for Ac.-α-tubulin (MCCs, blue), CC10 (Club cells, green), and Actin (cell membranes, magenta) show reduced MCC and Club cell numbers in RSPO2-treated cultures.

(B) RSPO2 does not reduce the number of ΔN-TP63+ (green) cells. Nuclei (DAPI, blue).

(C) Quantification from (A) and (B). Mann Whitney test, not significant, ns (p > 0.05); *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001.

(D) Quantitative real-time PCR. Expression levels are depicted relative to stage controls. RSPO2 reduces expression of MCC (FOXJ1, MCIDAS) and Club cell (SCGB1A1) markers but increases expression of BC markers (ΔN-TP63, KRT5). Student’s t test, not significant, ns (p > 0.05); *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001.

(E and F) Optical orthogonal sections of confocal images. RSPO2-treated cultures display increased epithelial thickness and cells staining for BC markers ΔN-TP63 (green, in E) and KRT5 (green, in F).

Related to Figure S4.