Figure 1.
KLF4 loss leads to change in number of goblet cells and morphology of the small intestinal epithelium.
(A) Left: IHC staining for Cre recombinase in Klf4−/− small intestine. Right: IHC staining for KLF4 in control and Klf4−/− small intestine tissues. (B) H&E staining of control and Klf4−/− small intestine tissues. (C) Small intestine treated with tamoxifen for 5 days were stained for Periodic acid-Schiff (PAS, top) and Alcian Blue (AB, bottom). (*, P<0.05) (D) Tissue slides from small intestine of control and Klf4−/− mice were stained for neurotensin (NT) antibody and detected by immunofluorescent antibody.
Figure 2.
KLF4 ablation leads to abnormal proliferation and differentiation in small intestinal epithelium.
(A) Small intestine from Klf4−/− mice induced by tamoxifen for different time endurances were stained by H&E and PAS, and also immunohistochemistry staining was performed with anti-Ki67, anti-Lysozyme, anti-DCAMKL-1, and anti-PCNA antibodies respectively. (B) Statistic analysis of IHC staining results from (A). (*, P<0.05) (C) IHC staining from (A) in higher magnification of highlighted frames. Bottom panel: IHC staining with ZO-1 antibody in one-month knockout intestine tissue.
Figure 3.
KLF4 is essential for cell polarity and crypt-cyst formation in 3D culture of Caco-2 cells.
(A) Immunofluorescent staining of Caco-2 cells in 2D and 3D culture with anti-α6-integrin and ZO-1 antibodies. (B) Caco-2 cells in 3D culture were stained for differentiation markers indicating cell polarity and cyst formation (definition of lumen-cyst vesus cyst structures). (C) Left: western blotting showing knockdown of KLF4 in Caco-2 cells. Right: statistical analysis of percentage of lumen-cyst formation in Caco-2 cell 3D cultures, comparing between control and KLF4 siRNA-transfected cell cultures. (*, P<0.05) (D) Left: western blotting showing expression of KLF4 in 293T cells co-transfected with human KLF4 and KLF4 shRNA plasmids. Right: statistical analysis of percentage of lumen-cyst formation in Caco-2 cell 3D cultures, comparing between control and KLF4 shRNA-infected cell cultures. (*, P<0.05).
Figure 4.
KLF4 facilitates cell polarity and crypt-cyst formation in colon cancer cells.
(A) LS174 cells in 3D culture system were stained for differentiation markers indicating cell polarity and cyst formation. (B) Statistical analysis of percentage of cyst formation in LS174T cell 3D cultures, comparing between doxycycline (Dox)-induced and non-induced cells.(*, P<0.05) (C) Western blotting indicating expression of KLF4 in LS174T cell line with or without induction of doxycycline. (D) Semi-quantitative RT-PCR showing expression of genes related to KLF4-regulated cell polarity and related to Wnt signaling.