Figure 1.
The Kaplan-Meier curve for overall survival rate of patients with type 1 EMCA (n = 120) according to the nuclear expression of YAP.
Increased nuclear immunoreactivity of YAP was significantly associated with worse overall survival (P = 0.015, log-rank test).
Table 1.
Comparison of demographic data and YAP expression between type 1 and type 2 EMCA.
Table 2.
Association of YAP expression in nucleus with clinicopathological factors in type1.
Figure 2.
(A) Expression of YAP and phospho-YAP (Ser127) in three EMCA cell lines by western blotting. (B) Immunofluorescent cytochemical staining of endogenous YAP using anti-YAP antibody (YAP: green, DAPI: blue).
Figure 3.
(A) Inhibited protein expression of YAP by YAP-specific siRNA (siYAP) was confirmed by western blotting both 72 and 96 hours after transfection (left). The number of viable cells between 24–96 hours after transfection of siYAP or control siRNA (siCont) was evaluated in HEC-1-B cells using the water-soluble tetrazolium salt assay (Cell counting kit-8) (right). 26.2% and 42.9% inhibition of cell proliferation were confirmed by knockdown of YAP expression at 72 and 96 hours, respectively. (* P<0.05 (the Mann-Whitney u-test)). Results are shown in means ±standard deviations (bars) in quadruplicate experiments. Similar trends were obtained in other two independent experiments. (B) The number of colonies in soft agar was compared between the YAP inhibited cells and the control cells to evaluate anchorage-independent cell growth in HEC-1-B cells. Representative image of formed colonies in soft agar (left). Quantitative analysis in number of formed colonies (right), showing a significant decrease in siYAP transfected cells. (* P<0.05 (the Mann-Whitney u-test)). Columns and bars represent means and standard deviation in sextuplicate experiments. Similar trends were obtained in another independent experiment. (C) Transwell migration and invasion assay. Representative image of migrated/invaded HEC-1-B cells (left). Quantitative analysis in number of migrated/invaded cells (right), which showed a significant decrease in siYAP transfected cells. (* P<0.05 (the Mann-Whitney u-test)). Columns and bars represent means and standard deviation in quadruplicate experiments. Similar trends were obtained in another independent experiment. (D) Flow cytometric analysis. Representative results of the population in each phase of the cell cycle in HEC-1-B EMCA cells 72 and 96 hours after transfection with siCont/siYAP (left). Quantitative analysis in the population in each phase (right). siYAP transfected cells showed a significant accumulation of cells in G0/G1 phase and significant decreases in S and G2/M phases at 72 and 96 hours (* P<0.05, ** P<0.001, the Student's t- test). Columns and bars represent means and standard deviation in triplicate experiments. Similar trends were obtained in another independent experiment.
Figure 4.
(A) Endogenous expression of YAP (approximately 65 kDa) and exogenous expression of YAP tagged with GFP protein (approximately 92 kDa in total) in HEC-1-A cells transfected with wild-type YAP (WT-YAP) and S127A mutant YAP (S127A-YAP). (B) Representative image of colony formation assay (left). Cells were transiently transfected with empty vector (EV)/WT-YAP/S127A-YAP and selected with appropriate concentrations of G418 for four weeks. The drug-resistant colonies formed by the YAP-transfected cells were significantly numerous compared with the control (the Mann-Whitney U-test). Columns and bars represent means and standard deviation in quadruplicate experiments. Similar trends were obtained in another independent experiment.
Figure 5.
Clonogenic assay in HEC-1-B cells after radiation exposure.
Knockdown of YAP expression by siRNA reduced clonogenic survival in HEC-1-B cells, resulting in an increase in radiation sensitivity with a dose enhancement factor at 10% survival (DEF 0.1) of 1.36. Results are shown in means ±standard deviations (bars) in triplicate experiments. Similar trends were obtained in other three independent experiments.