Fig 2: Twist1 positively regulates Claudin-4 expression. Transfection of HeLa and SiHa cells with 20 nM siRNA targeting Twist1 or scrambled control siRNA was performed. Briefly, 48 h after transfection, HeLa (A) and SiHa (B) cells were subjected to western blotting. Transfection of HeLa and SiHa cells with 20 nM siRNA targeting Twist1 or scrambled control siRNA was performed. 48 h after transfection, HeLa (C) and SiHa (D) cells were subjected to RT-qPCR analysis. *P<0.05, **P<0.01 vs. siCtrl. Transfection of 20 nM siRNA targeting Twist1 or scrambled control siRNA into (E) HeLa and (F) SiHa cells. Co-transfection with carrier or an overexpression plasmid for Twist1 was performed 24 h after transfection. Cells were collected 48 h after transfection and examined by western blotting using specific antibodies. After treatment, (G) HeLa and (H) SiHa cells were collected 48 h after transfection and analyzed by RT-qPCR. *P<0.05, **P<0.01 vs. vector. RT-q, reverse transcription-quantitative PCR; siRNA, small interfering RNA; Ctrl, control.

Fig 3: TGF-β induces Claudin-4 expression by upregulating Twist1. Treatment of (A) HeLa and (B) SiHa cells with TGF-β for 48 h, and whole-cell extracts were harvested for western blotting using specific antibodies. (C) HeLa and (D) SiHa cells with E-Box1 of Claudin-4 deletion using the CRISPR-Cas9 system were treated with TGF-β for 48 h, and whole-cell extracts were harvested for western blotting using specific antibodies. (E) Wild-type HeLa and (F) E-Box1-deleted HeLa cells were exposed to TGF-β for 48 h before RT-qPCR analysis of Twist1 and Claudin-4. **P<0.01, ***P<0.001 vs. control. A total of 20 nM siRNA against Twist1 or scrambled control siRNA was transfected into HeLa and SiHa cells. Cells were treated with TGF-β. (G) HeLa and (H) SiHa cells were subsequently harvested for immunoblotting with specific antibodies. (I) Following treatment, HeLa cells were subsequently harvested for RT-qPCR analysis of Twist1 and Claudin-4. **P<0.01, ***P<0.001 vs. siCtrl or siCtrl + TGF- β. RT-q, reverse transcription-quantitative; siRNA, small interfering RNA; Ctrl, control; ns, not significant.

Fig 4: Twist1 upregulates Claudin-4 transcription via E-Boxes in the Claudin-4 promoter. (A) Potential Twist1 consensus binding E-Boxes sites in the Claudin-4 promoter region. E-Box1 and E-Box2 sites upstream of the start codon of Claudin-4 are shown. The constructed deletion mutants of the Claudin-4 promoter (−1,500-2,200del and −206-1,500del) and Claudin-4 full-length promoter reporter constructs are presented. Transfection of (B) HeLa and (C) SiHa cells with 20 nM siRNA targeting Twist1 or scrambled control siRNA was performed. Plasmids expressing pGL3 or pGL3 carrying the Claudin-4 promoter (Claudin-4-Luc) with two potential Twist1 binding E-Box domains and Renilla luciferase were co-transfected. Luciferase activity was measured after 24 h using the dual-luciferase reporter assay system and normalized to Renilla luciferase activity. **P<0.01, ***P<0.001 vs siCtrl. (D) HeLa and (E) SiHa cells containing co-expression of plasmids that express Renilla luciferase and pGL3 or pGL3 bearing the Claudin-4 promoter with two potential Twist1-binding E-Box sites (WT), with the ablation of E-Box2 (Claudin-4 Luc E-Box1) or with the ablation of E-Box1 (Claudin-4 Luc E-Box2), followed by dual-luciferase reporter assay. ***P<0.001 vs. Claudin-4 Luc WT. (F) Control or an overexpression plasmid of Twist1 was transfected into HeLa cells. Co-transfection with Claudin-4 Luc WT, Claudin-4 Luc E-Box1, or Claudin-4 Luc E-Box2 plasmids was performed 24 h after transfection. Luciferase activity was measured after another 24 h. Fresh HeLa cells were harvested and lysed for chromatin immunoprecipitation assay with normal IgG or anti-Twist1 antibody. *P<0.05 vs. vector. (G) Promoter fragments were amplified by PCR utilizing primers specific for putative E-Box on the Claudin-4 promoter region or GAPDH (negative control). (H) Reverse transcription-quantitative PCR was performed to quantify the relative enrichment of Twist1 at potential Twist1-binding E-Box domains. ***P<0.001 vs. IgG. siRNA, small interfering RNA; siCtrl, scrambled control siRNA; WT, wild-type; del, deletion; ns, not significant.

Fig 5: Claudin-4 knockdown suppresses cell migration and invasion caused by Twist1. (A) Specific siRNA (20 nM) was transfected into HeLa cells. Co-transfection with vector or a Twist1-overexpressed plasmid was performed on cells 24 h after transfection, and 12–24 h later, cells were assessed by Transwell migration assay. (B) Cells that could migrate were counted in three randomly selected fields of view/chamber insert. (C) After treatment, HeLa cells were assessed by Transwell invasion assay. (D) Cells that could invade were counted in three randomly selected fields of view/chamber insert. *P<0.05, **P<0.01, ***P<0.001 vs. siCtrl + vector or siClaudin-4 + vector. (E) Cell lysates were collected, and efficiency of Claudin-4 knockdown or Twist1 overexpression was assessed by western blot analysis. siRNA, small interfering RNA; siCtrl, scrambled control siRNA.