FIGURE 4.

Hippo signalling is essential for EHD1‐mediated enhancement of stemness in vitro and in vivo. (A) Western blot analysis validated the effect of treatment with VP (1 μM) or DMSO for 24 h on the Hippo signalling pathway in the EHD1^KD+WT group. The expression of core components and downstream targets of the Hippo signalling pathway was detected. (B‐C) The 3D spheroid cancer models detected the effect of VP treatment on stemness in the EHD1^KD+WT group. The bar graphs show the quantification of the number of spheres per well formed by A549‐derived cells. (D) The stemness of A549 cells treated with DMSO or VP was evaluated by clonal heterogeneity analysis. (E) Expression of stemness‐related proteins in A549‐derived cells treated with DMSO or VP. (F‐H) Hippo signalling is critical for the EHD1‐mediated enhancement of A549 cell stemness in vivo. (F) Photographs showing the efficiency of tumour generation or tumoursphere formation in nude mice (upper panels). The panels below the images show the bioluminescence signal intensities in nude mice injected subcutaneously with EHD1^KD+Ctrl (left) or EHD1^KD+WT (right). (G) Images showing the efficiency of tumour generation or tumoursphere formation in nude mice treated with DMSO or VP (upper panels). Bioluminescence signal intensities in nude mice injected subcutaneously with EHD1^KD+WT and treated with DMSO or VP are shown (lower panels). (H) The stemness of A549 cells was estimated as the stem cell frequency, with upper and lower 95% confidence intervals. The tumour engraftment efficiency in vivo was successfully reduced by VP treatment. The data are shown as the mean ± SD values. p > .05 was considered N.S., *p < .05, **p < .01 and ***p < .001