Figure 5

NANOG promotes EMT in epithelial cells

. (A) Immunoblot of NANOG and P63 in nuclear extracts from HaCat cells transduced with a human NANOG expressing vector (HaCat/NANOG1) or empty vector control (HaCat/EV). NTERA2 teratocarcinoma cells were used as a positive control for NANOG, P63 was used as a keratinocyte marker and SMC-1 was used as a loading control. (B) Relative mRNA levels of the indicated genes in HaCat cells treated as in (A). Samples were analyzed after puromycin selection. mRNA levels were normalized by GAPDH levels and then expressed as the ratios with respect to the levels in HaCat/EV (shown as fold change). Values correspond to two independent biological replicates (n = 2). (C) Percentage of migration of HaCat cells transduced with NANOG1 or empty vector (n = 3). Areas were measured as percentage of migrated distance during a 24hr period (measured across the scratch wound width). (D) Immunoblots of NANOG using nuclear lysates of TE2 (human esophageal SCC cell line) cells transfected with pools of scrambled (siRNA SCR) or anti-NANOG (siRNA NANOG) siRNAs. Samples were analyzed 48 hr after transfection. SMC-1 was used as a loading control. L.E refers to low exposure of the film after incubation with ECL. H.E. refers to high exposure of the film after incubation with ECL. (E) Relative mRNA levels of the indicated genes in TE2 cells treated as in (D). mRNA levels were normalized by GAPDH levels and then expressed as the ratios with respect to the levels in TE2/siRNA SCR (shown as fold change). Values correspond to two transfections (n = 2). Bars in (B,C,E) correspond to mean ± SD. Statistical significance was determined by the two-tailed Student’s t test: n.s., non significant; (*) P < 0.05; (**) P < 0.01; (***) P < 0.001.