NEDD9 is required for maintenance of a stem-like phenotype. (A, E) Expression of NEDD9 was confirmed between NEDD9 knockdown/overexpression and vector cells. (B, F) The capability of tumor sphere formation was measured between NEDD9 knockdown/overexpression and vector cells. Scale bar = 100 μm. (C, G) Expression of stemness-related transcription factors, KLF4 and ALDH1A3, was detected between NEDD9 knockdown/overexpression and vector cells. (D, H) Expression of stemness markers, CD90 and CD271, was analyzed by flow cytometry between NEDD9 knockdown/overexpression and vector cells. (I) Cells were dissociated into 1, 100, and 1,000 cells for 7 days of suspension culture. Scale bar = 100 μm. (J) Tumorigenic cell frequency in each fraction of ESCC cells was determined by a limiting dilution assay in NOD/SCID mice. *P < 0.05; **P < 0.01; ***P < 0.001.