Fig. 7

TGFβ downregulates SGO1-AS1 transcription via ZEB1. a. qRT-PCR analyses of the SGO1-AS1, SNAI and ZEB1 levels in SGC7901 cells incubated with TGFβ1 (3 ng/mL or 10 ng/mL) for 24 h. b. Expression levels of SGO1-AS1, SNAI and ZEB1 in BGC823 and AGS cells stimulated with 10 ng/mL TGFβ1 for 24 h. *P < 0.05, **P < 0.01 and ***P < 0.001 compared to those without TGFβ1 stimulation. c. SGO1-AS1, SNAI and ZEB1 levels in SGC7901 cells incubated with SB431542 (10 μM) for 24 h. *P < 0.05, **P < 0.01. d. A schematic diagram illustrating the four putative ZEB1 binding sites (Sites A, B, C and D) in the SGO1-AS1 promoter. e. Luciferase reporter assays of the SGO1-AS1 promoter region containing either wild-type (WT) or mutated (Mut A, Mut B, Mut C, Mut D, or Mut A-D) ZEB1 binding sites without or with TGFβ1 exposure. *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant compared to without the TGFβ1 stimulation. f. SGO1-AS1 expression was examined by a qRT-PCR analysis in TGFβ1-treated SGC7901 and BGC823 cells transfected with ZEB1 siRNAs. Western blot analysis was performed to assess the inhibition efficiency in the same cells (right). **P < 0.01. g. Upper: Putative ZEB1-binding sites on the SGO1-AS1 promoter region and design-indicated primers. Lower: ChIP analysis of ZEB1 enrichment on the SGO1-AS1 promoter in SGC7901 cells treated with TGFβ1. IgG and anti-GAPDH antibodies were used as controls. *P < 0.05, ***P < 0.001. In all cases, error bars indicateSDs from three independent experiments