Fig. 4

miR-375 downregulated ENAH, thus repressing proliferation, invasion, migration and stemness, and promoting apoptosis of KYSE70 cells in vitro. A, The mRNA expression of ENAH was determined by RT-qPCR in KYSE70 cells, relative to U6 or GAPDH. B, The protein expression of ENAH was determined by western blot analysis in KYSE70 cells, relative to U6 or GAPDH. C, Proliferation of KYSE70 cells in response to inhibition of both ENAH and miR-375 or either alone, as assessed by EdU assay (scale bar = 50 μm). D, Invasion and migration of KYSE70 cells in response to inhibition of both ENAH and miR-375 or either alone, as assessed by Transwell assay (scale bar = 50 μm). E, Tumorsphere formation of KYSE70 cells in response to inhibition of both ENAH and miR-375 or either alone, as assessed by tumorsphere formation assay (scale bar = 100 μm). F, Apoptosis of KYSE70 cells in response to inhibition of both ENAH and miR-375 or either alone, as assessed by flow cytometry. G, The mRNA expression of Bcl-2, Bcl-xl, Bax, CD133, Nanog and OCT-4 was measured by RT-qPCR in KYSE70 cells, relative to GAPDH. H, Representative western blots of E-cadherin, N-cadherin, Snail, Bcl-2, Bcl-xl, Bax, CD133, Nanog and OCT-4 proteins and their quantitation in KYSE70 cells, relative to GAPDH. * p < 0.05 vs. KYSE70 cells transfected with siRNA-NC by one-way ANOVA. Data are shown as mean ± standard deviation of three technical replicates