Figure 1.
KLF4 inhibits cell proliferation and colony formation.
A. Cell proliferation of SKOV3 cells transduced with either EGFP or KLF4 was examined by MTT assay at different time points following Dox treatment. Overexpression of KLF4 significantly reduced cell proliferation compared to that in Dox-treated control cells (*p<0.05). B. 200 SKOV3 cells transduced with lentiviral KLF4 or EGFP control vectors were seeded into each well of a 6-well plate and cultured for 14 d. Cell colonies were counted following crystal violet staining. The number of colonies in KLF4-overexpressing cells was significantly reduced compared to that in Dox-treated control cells (***p<0.001). C. The number of colonies in KLF4-overexpressing OVCAR3 cells was significantly reduced compared to that in Dox-treated control cells (**p<0.01). D. Soft agar colony formation assay was performed in triplicate using SKOV3 cells. Colonies were photographed and counted after 3 weeks. The number of colonies in KLF4-overexpressing cells was significantly reduced compared to that in Dox-treated control cells (***p<0.001).
Figure 2.
KLF4 reduces cell migration in ovarian cancer cells.
A. Wound-healing assay was performed to examine the migration rate of SKOV3 cells transduced with KLF4 and EGFP lentiviral vectors. Photographs were taken at 0 and 24 h following the initial scratch. Migration rates were quantified by measuring three different wound areas. Three separate experiments were performed. Migration rate was significantly reduced in KLF4-overexpressing cells compared to that in Dox-treated controls (**p<0.01). B, C. Transwell migration assay was performed in SKOV3 and OVCAR3 cells. Overexpression of KLF4 significantly reduced cell migration in SKOV3 (B) and OVCAR3 (C) cells compared with that in Dox-treated controls (***p<0.001).
Figure 3.
KLF4 reduces ovarian cancer cell invasion.
A, B. Cell invasion assay was performed using Matrigel-coated transwell plates for SKOV3 (A) and OVCAR3 cells (B). The invasion rate was significantly reduced in KLF4-overexpressing cells compared to that in Dox-treated control cells from both SKOV3 and OVCAR3 cells (**p<0.01). Data were collected from three separate experiments and analyzed using Student t-tests.
Figure 4.
KLF4 promotes mesenchymal-epithelial cell transition.
A. B. Western blots were performed in KLF4- and EGFP-transduced SKOV3 (A) and OVCAR3 cells (B) with or without Dox induction. E-cadherin expression was significantly upregulated (***p<0.001), whereas vimentin (**p<0.01) and snail2 (***p<0.001) were downregulated in KLF4- overexpressing SKOV3 cells compared to control cells (A). E-cadherin (**p<0.01) was upregulated, whereas vimentin (*p<0.05) and snail2 (***p<0.001) were downregulated in KLF4-overexpressing OVCAR3 cells compared to Dox-treated control cells (B). E-cadherin (C) and vimentin (D) were immunostained in cellular membranes in KLF4-expressing and control SKOV3 cells. E. Snail2 was stained in cell nuclei in KLF4-expressing and control SKOV3 cells. F. KLF4 binding to the promoter of E-cadherin in SKOV3 cells was examined by chromatin immunoprecipitation using KLF4 antibody and detected by real-time PCR using E-cadherin-specific primers. The ChIP-enriched DNA levels were normalized to input DNA, followed by subtraction of non-specific binding determined by control IgG (***p<0.001).
Figure 5.
TGFβ promotes EMT in ovarian cancer cells.
Human ovarian cancer cell lines SKOV3 (A) and OVCAR3 (B) were treated with different doses of TGFβ for 48 h. EMT-associated marker genes, including E-cadherin, snail2, and vimentin were examined using Western blot. Significances were determined by comparing TGFβ treated to non-treatment (*p<0.05, **p<0.01, ***p<0.001).
Figure 6.
KLF4 inhibits TGFβ-induced EMT in ovarian cancer cells.
Ovarian cancer cell lines SKOV3 (A) and OVCAR3 (B) transduced with lentiviral KLF4 overexpression and control vector were treated with TGFβ for 48 h, and the protein expressions of EMT-associated marker genes E-cadherin, snail2 and vimentin were examined using Western blot. Significant differences were compared between KLF4 expressing and control group (**p<0.05, ***p<0.001).