Fig 2: A schematic diagram depicting the TGF-β/Smad-GTSE1-SNAIL1 signaling axis in Huh7 cells. GTSE1, G2 and S phase-expressed-1; SNAIL1, snail family transcriptional repressor 1; LMB, leptomycin B; SBEs, Smad binding elements.

Fig 3: TGF-βI-induced migratory ability of Huh7 cells is dependent on GTSE1 expression. (A) Transwell assay of Huh7 cells after GTSE1 knockdown with or without TGF-βI treatment. Left panel: Representative images of three independent assays. Scale bar, 50 µm. Right panel: Quantification of the migrated cells per field for each group (n=3). *P<0.05, **P<0.01, ***P<0.001 and NS vs. siNC group. (B) Transwell assay of Huh7 cells with GTSE1 overexpression. Left panel: Representative images of three independent assays. Scale bar, 50 µm. Right panel: Quantification of the migrated cells per field for each group (n=3). **P<0.01 vs. Vector group. NS, not significant; GTSE1, G2 and S phase-expressed-1; NC, negative control; si, small interfering RNA.

Fig 4: GTSE1 increases SNAIL1 protein turnover via a proteasome-dependent pathway. (A) Endogenous expression of SNAIL1 was detected via western blotting in Huh7 cells transfected with an increasing concentration of GTSE1 plasmid, and co-treatment using the proteasome inhibitor MG-132 (10 µM) for an additional 12 h at 37°C before harvesting as indicated. The arrowhead indicates a non-specific band. Semi-quantitative data of optical band densitometry are shown. The data are presented as the mean ± SD (n=3). ***P<0.001 vs. lane 2 for the fold-change of HA; *P<0.05 vs. lane 1; ###P<0.001 vs. lane 3 for the fold-change of SNAIL1. Protein turnover of endogenous expression of SNAIL1 after GTSE1 (B) overexpression or (C) knockdown over the course of 2 h following the addition of 10 µg/ml CHX. Tubulin was used as a loading control. Images are representative of three independent experiments and fold-changes of SNAIL1 are shown below by densitometry, which was normalized to controls. The data are presented as the mean ± SD (n=3). *P<0.05, **P<0.01, ***P<0.001 vs. Vector group or siNC group at each time point. GTSE1, G2 and S phase-expressed-1; SNAIL1, snail family transcriptional repressor 1; NC, negative control; si, small interfering RNA; CHX, cycloheximide; HA, human influenza hemagglutinin epitope.

Fig 5: TGF-βI induces the expression of GTSE1 via SBEs in the promoter region. (A) Endogenous protein expression levels of both GTSE1 and SNAIL1 were detected via western blotting with increasing concentrations of TGF-βI treatment for 24 h at 37°C in Huh7 cells. Semi-quantitative data of optical band densitometry are shown. The data are presented as the mean ± SD (n=3). ***P<0.001 vs. control group. (B) GTSE1 and SNAIL1 mRNA expression levels were detected via RT-qPCR after TGF-βI treatment (20 ng/ml) for 24 h at 37°C in Huh7 cells. GAPDH was used as an internal control. Data are expressed as the mean ± SD (n=3). ***P<0.001 and NS vs. non-treated group. (C) Schematic representation of pGL3-luc reporter constructs of the GTSE1 promoter (from −1,500 to TSS) with or without deletion of potential Smad binding elements (squares) as indicated. (D) RLU of different GTSE1 promoter constructs was determined using a luciferase reporter assay. Huh7 cells were co-transfected with the indicated pGL3-luc reporter constructs and Renilla luciferase. Then, 1 day after transfection, cells were treated with 0 or 20 ng/ml TGF-βI for an additional 24 h at 37°C. Data are expressed as the mean ± SD (n=3). ***P<0.001 and NS vs. pGL3-GTSE1-PM group. (E) Endogenous expression of SNAIL1 was detected via western blotting after GTSE1 depletion with or without TGF-βI treatment in Huh7 cells. Semi-quantitative data of optical band densitometry are shown. The data are presented as the mean ± SD (n=3). **P<0.01, ***P<0.001 and NS, not significant vs. siNC group. (F) GTSE1 and SNAIL1 mRNA expression levels were detected via RT-qPCR after GTSE1 knockdown with or without TGF-βI treatment in Huh7 cells. Data are expressed as the mean ± SD (n=3). *P<0.05, ***P<0.001 and NS vs. siNC group. NS, not significant; RLU, relative luciferase activity; GTSE1, G2 and S phase-expressed-1; SNAIL1, snail family transcriptional repressor 1; NC, negative control; si, small interfering RNA; TSS, transcription start site; DEL, deletion; PM, promoter.