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Fig. 3 | Journal of Experimental & Clinical Cancer Research

Fig. 3

From: Inactivation of the tight junction gene CLDN11 by aberrant hypermethylation modulates tubulins polymerization and promotes cell migration in nasopharyngeal carcinoma

Fig. 3

Methylation inhibits CLDN11 promoter activity by impairing the binding of the transcriptional activator GATA1. a Different promoter activities of a series of CLDN11 promoter deletion luciferase reporters were assayed in TW02 and HK1 cells. Intergroup comparison was conducted relative to the empty vector (pGL3). b The minimal CLDN11 promoter reporter (− 213 to + 197) and the FLAG-tagged GATA1 and GATA2 expression clones were cotransfected into TW02 and HK1 cells. Three putative GATA binding sites: GATA1 (− 92), GATA1/2 (− 62), and GATA2 (+ 184), are indicated. GATA1: GATA1 overexpression, GATA2: GATA2 overexpression, GATA1 + 2: GATA1 and GATA2 co-overexpression. Intergroup comparison was conducted relative to the vector control. The expression levels of ectopic GATA-1, GATA-2, and actin (internal control) were examined through Western blotting. c Minimal CLDN11 promoter reporters with wild-type (WT) and three mutated GATA binding sites (MU, S1–S3) were transfected into TW02 and HK1 cells, respectively. Intergroup comparison was conducted relative to the empty vector (pGL3). a~c All the luciferase reporter activities were normalized with respect to renilla activity. d DNA pull-down assay was used to analyze the binding affinity of exogenous FLAG-tagged GATAs (GATA1–3F and GATA2-3F) to WT, methylated (ME), and mutated (MT) biotinylated probes containing GATA1/2 site; the sequences of these probes are shown in the upper panel. Methylated cytosine is indicated by using “m” above C, and mutated sequences are underlined. Anti-GATA1 and anti-FLAG antibodies were used for examining the amount of bound GATA1–3F and GATA2-3F in the immunoprecipitates and 5% input. e EMSA was performed to compare the binding affinity of purified recombinant GATA1 for WT, ME, and MT biotinylated probes of the GATA1/2 site. The arrow indicates DNA–protein complexes. Antibodies against GATA1 were used for supershift assays

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