Figure 4

GLUT3 is a direct ZEB1-target gene. (A) The indicated cell lines were lysed with RIPA buffer to prepare protein extracts and to analyze the expression of ZEB1, or β-tubulin as control, by Western blot. (B) IGV Browser view of 33 kb around the SLC2A3 gene including ZEB1 (black), RNA Polymerase II (RNAPolII, green), the active histone mark H3K27ac (green-yellow) ChIP-seq, and Input (grey) tracks in human lymphoblastoid cell lines. ZEB1 peak calls and ‘CACCTC’ sequence matches on the reverse strand are also indicated (black). The orientation of SLC2A3 is indicated by an arrow. (C) Extracts from SW1573 cells were used for ZEB1 ChIP followed by real-time PCR. The coordinates of the PCR amplicons relative to the TSS are indicated. CDH1 and an untranscribed sequence upstream of GAPDH were used as positive and negative controls for ZEB1 binding, respectively. The data shown are representative of three independent experiments. (D) (Upper part) scheme showing the promoter region of 1,000 bp preceding the transcription start site (prom.) and the intron 2 region (int.2), which were amplified from the SLC2A3 gene and cloned into the luciferase reporter construct. Ex. exon, LUC luciferase. (Lower part) 293 T cells were transfected with the indicated luciferase reporter plasmids. Luciferase activity was analyzed 24 h later. Data show means ± s.d. (n = 5) of relative luciferase activity. (Lower right) 293 T cells were transfected with control (ctl) or ZEB1 siRNAs and were lysed 96 h later to prepare protein extracts and to analyze the expression of ZEB1, or β-tubulin as control, by Western blot. (E) 293 T cells were transfected with the indicated luciferase reporter plasmids. For the int.2 construct, either wild-type (WT) or a variant with a deletion of the E-box-like motif (Δ-CACCTC) was used. Luciferase activity was analyzed 24 h later. Data show means ± s.d. (n = 5) of relative luciferase activity.