PMC

Knock-down of SALL4 inhibits tumor formation in xenograft models in vivo. a Tumor volumes in mice injected with shSALL4 TE7 cells were remarkably smaller than the scramble group. Both tumor incidence (b) and tumor weight (c) were decreased in mice transplanted with shSALL4 TE7 cells compared with those injected with scramble cells. Tumor growth (d) was inhibited in mice transplanted with shSALL4 TE7 cells. (**P < 0.01)

Expression of SALL4 in ESCC tissues. a and b Real-time PCR analysis of SALL4 mRNA expression in ESCC (T) and paired non-cancerous (N) tissues (***P < 0.001). c Representative photomicrographs of immunohistochemical staining for SALL4 in ESCC tissues and adjacent normal tissues. 1-Negative staining, 2-Normal tissues, 3-Weak staining, 4-Strong staining. d The Kaplan-Meier curves for the overall survival in ESCC patients with SALL4-low and SALL4-high expression (P = 0.0027). Original magnification: 400×

Down-regulation of SALL4 promotes mesenchymal-epithelial transition (MET) via Wnt/β-catenin signaling pathway during ESCC tumorigenisis. The expression of epithelial cell marker (E-cadherin) was elevated, while mesenchymal cell marker (Vimentin) was decreased in TE7 shSALL4 cells compared with scramble cells both in mRNA level (a) and protein level (b). The expression of the key molecules in Wnt/β-catenin pathway Wnt3a and β-catenin was down-regulated determined by real-time PCR (c) and western blotting (d) after silencing of SALL4. (**P < 0.01, ***P < 0.001)

Silencing of SALL4 suppresses migration and invasion capabilities in vitro. a The migratory and invasive capabilities of TE7 and EC109 cells were evaluated by using transwell migration and invasion assay. Representative microscopic images of the bottom chamber were shown. b The bar graphs represented the average number of migrated cells and invaded cells on the underside of the membrane. The cell colony was decreased after transfection in both shSALL4 TE7 (c) and EC109 (d) cells. Colonies with >100 cells were quantified. Original magnification: 200×. (*P < 0.05, **P < 0.01, ***P < 0.001)

SALL4 is necessary for the maintenance of CSC properties. a Bright-field microscopy images of spheres generated from scramble and shSALL4 TE7 cells after the sphere-formation assay. Original magnification: 200×. b The expression of stemness-related markers (Sox2, Oct4, Nanog) was down-regulated in shSALL4 cells compared with scramble cells in TE7. c The expression of CSC markers SALL4, CD44, CD133 and ALDH1 were up-regulated in TE7 spheres than control adherent cells. d Real-time PCR showing CD44, CD133 and ALDH1 in spheres generated from scramble and shSALL4 TE7 cells. e The sensitivity of shSALL4 TE7 cells to cisplatin was increased in a concentration-dependent manner compared with scramble cells. f The apoptosis rate was enhanced in shSALL4 TE7 cells treated with 10 μg/mL cisplatin. (*P < 0.05, **P < 0.01, ***P < 0.001)

Silencing of SALL4 inhibits cell proliferation, induces apoptosis and arrests cell cycle in vitro. a Real-time PCR analysis of SALL4 expression in Het1A, TE1, TE7, EC1, EC109, EC9706, KYSE70, KYSE450 cell lines. b The mRNA level of SALL4 was verified in sorted TE7 and EC109 cells after transfection. c The protein level of SALL4 in sorted TE7 and EC109 cells was assessed by using Western blotting. β-actin was used as an internal control. d Cell viability was evaluated at indicated time points using CCK8 assay. e Cell apoptosis was measured by flow cytometric analysis. f Knock-down of SALL4 induced cell cycle arrest at G0/G1 phase. (*P < 0.05, **P < 0.01, ***P < 0.001)