Fig. 2

5-aza-dC application induces CLDN6 expression and inhibits migration and invasion abilities in MCF-7 cells. a RT-PCR was used to display the effect of 5-aza-dC on CLDN6 expression. 5-aza-dC application induced CLDN6 expression in a concentration- and time-dependent manner. Lane 1 was control group; lanes 2 to 6 were treated with 5-aza-dC group (2: 2.5 μM; 3: 5 μM; 4: 10 μM; 5: 15 μM; 6: 20 μM). Data in lane 6 showed that 20 μM 5-aza-dC treatment had the greatest effect on inducing CLDN6 expression. b CLDN6 expression was determined in MCF-7 cells treated with 20 μM 5-aza-dC for 1 to 72 h. Lane 1 was control group, lane 2 to 5 were 20 μM 5-aza-dC treated group (2: 12 h; 3: 24 h; 4: 48 h; 5: 72 h). Data in lane 5 showed that 20 μM 5-aza-dC for 48 h had the greatest effect on inducing CLDN6 expression. c CLDN6 protein expression was significantly up-regulated in MCF-7 cells treated with 5-aza-dC at 20 μM for 48 h by western blot. d Immunofluorescent staining showed CLDN6 expression at the membranes of MCF-7 cells after treated with 20 μM 5-aza-dC for 48 h using × 100 magnification. e CLDN6 DNA methylation status was determined by MSP analysis between control group and treatment with 5-aza-dC 20 μM for 48 h group. 5-aza-dC treating group increased demethylation of CLDN6 CpG sites. f MCF-7 cells treated with 5-aza-dC were examined the ability of cell migration compared with control by wound-healing assay. g The invasive cells treated with 5-aza-dC were remarkably lower invasive ability than control group by invasion assay. *P < 0.05. CLDN6, claudin-6; 5-aza-dC, 5-Aza-2’-deoxycytidine; MSP, Methylation-Specific PCR