Figure 4.

Slug downregulates the expression of βIII-tubulin/TUBB3 and βIVa-tubulin/TUBB4. (A) The mRNA expression levels of TUBB, TUBB1, TUBB2B, TUBB3, and TUBB4 were determined using real-time RT-PCR in A549/EGFP, A549/Slug, Ma1/EGFP, Ma1/Slug, H1299/EGFP, and H1299/Slug cells. GAPD was used to normalize the expression levels. Rel mRNA: normalized mRNA expression levels (target genes/GAPD × 10³). The data shown represent the ratio of mRNA expression in Slug-overexpressing cells/EGFP cells with average ± SD of three independent experiments. TUBB, βI-tubulin isotypes; TUBB1, βVI-tubulin; TUBB2B, βII-tubulin; TUBB3, βIII-tubulin; TUBB4, βIVa-tubulin. (B) Western blot analysis for β-tubulin isotypes. βIII-tubulin and βIV-tubulin expression were suppressed in A549/Slug cells. β-actin was used as an internal control. (C) The TUBB4 promoter activity was examined using a luciferase reporter assay. Luciferase vectors with either an empty or a TUBB4 promoter (pGL4.14-mock or pGL4.14-TUBB4) that includes two E2-box sequences (E2-box #1 and E2-box #2) were transiently cotransfected with a mock or Slug expression plasmid (pcDNA3.1-mock or pcDNA3.1-Slug) expressing β-galactosidase as an internal control. The results were normalized to β-galactosidase activity and are representative of at least three independent experiments. *P < 0.05. (D) Chromatin immunoprecipitation (ChIP) of Slug on the promoter of TUBB4. A549/EGFP and A549/Slug cells were used for analysis. Two putative regions in the TUBB4 promoter that contain the E2-box sequences (E2#1 and E2#2) were amplified using PCR. Primers to the GAPDH promoter were used as the control. Inputs, 1% of chromatin used for ChIP; H₂O, no DNA templates. EGFP, enhanced green fluorescent protein; SD, standard deviation; PCR, polymerase chain reaction; mRNA, messenger ribonucleic acid.