Immunohistochemical staining of the rat intestinal mucosa fixed with Carnoy solution (original magnification ×400).

<p>Rat TFF3 (A), IgGFcγBP (B) and Muc2 (C) existed in the two mucus layers and goblet cells.</p

Micrographs of rat intestinal mucosa of the colon fixed with Carnoy solution and stained by AB/PAS staining method (original magnification ×400).

<p>Normal colonic mucosa contained two mucus layers, the top, loose layer of mucus (arrowhead) and a stratified, firm layer of mucus (arrow) (A); the colonic mucosa after suction of the mucus contained only the stratified firm layer of mucus (arrow) (B); the colonic mucosa after suction and scraping of the mucus (C).</p

Deoxycholic acid (DCA) confers an intestinal phenotype on esophageal squamous epithelium via induction of the stemness-associated reprogramming factors OCT4 and SOX2

<p>Barrett's esophagus (BE) is essentially a metaplasia in which the normal stratified squamous epithelium is replaced by columnar epithelium. This study focuses on the involvement of OCT4 and SOX2, 2 key cell-reprogramming factors, in the deoxycholic acid (DCA)-induced expression of the intestinal hallmarks Cdx2 and MUC2 using both in vivo and in vitro models. Up-regulated expression of OCT4 and down-regulated expression of SOX2 were observed in BE compared with normal esophagus and esophagitis. Consistent with the data in vivo, DCA induced time-dependent expression of OCT4 at both the mRNA and protein levels and decreased nuclear expression of SOX2 in Het-1A cells. Down-regulation of OCT4 expression by siRNA abrogated DCA-induced expression of Cdx2 and MUC2, whereas siRNA against SOX2 significantly upregulated the expression of both Cdx2 and MUC2. Our data indicate that both OCT4 and SOX2 play important roles in the development of BE triggered by bile acid reflux.</p

Coimmunoprecipitation of rat TFF3, IgGFcγBP or Muc2 in the non-reduced (A) and reduced (B) intestinal mucus and blotted with each antibody.

<p>6 kDa proteins were detected with the anti-rTFF3 antibody in the immunoprecipitates with anti-rTFF3, anti-rIgGFcγBP and anti-Muc2 antibodies. 164 kDa proteins were detected with the anti-rIgGFcγBP antibody in the immunoprecipitates with anti-rTFF3, anti-rIgGFcγBP and anti-rMuc2 antibodies. The 140 kDa proteins were detected with the anti-rMuc2 antibody in the immunoprecipitates with anti-rTFF3, anti-rIgGFcγBP and anti-rMuc2 antibodies.</p

Transfection of miR-302b mimic in shRNA-Ascl2/HT-29 cells restores their ‘stemness’ characteristics and recovers their cellular behaviors in vitro.

<p>The let-7b, miRNA-124 miRNA-125b are significantly up-regulated, the miRNA-302b, miRNA-20a and miRNA-17 are significantly down-regulated, in shRNA-Ascl2/HT-29 cells compared with shRNA-Ctr/HT-29 cells (*: p<0.05;**: p<0.01) (A). The native shRNA-Ascl2/HT-29 cells, shRNA-Ascl2/HT-29 cells transfected with NC mimic and shRNA-Ascl2/HT-29 cells transfected with miR-302b mimic were analyzed for tumorsphere formation and were quantified at 100× (left panel of B) and 400× (right panel of B) magnifications. The number of tumorspheres and cells per tumorsphere from shRNA-Ascl2/HT-29 cells transfected with miR-302b mimic were significantly higher than those from shRNA-Ascl2/HT-29 and shRNA-Ascl2/HT-29 cells transfected with NC mimic (*: p<0.05) (C). Western blot analysis of Ascl2, C-myc, CD133, Bmi1, Sox2 and Oct4 in the cell lysates indicate that Ascl2, Sox2 and Oct4 protein levels are induced due to miR-302b mimic transfection in shRNA-Ascl2/HT-29 cells compared with shRNA-Ascl2/HT-29 and shRNA-Ascl2/HT-29 cells transfected with NC mimic (D). The real time PCR experiments for quantification of Ascl2, Oct4 and Sox2 mRNAs demonstrate a significant increase due to miR-302b mimic transfection of shRNA-Ascl2/HT-29 cells (*: p<0.05) (E). The colony-forming ability, the numbers of invaded cells and migrated cells of shRNA-Ascl2/HT-29 cells transfected with miR-302b mimic were significantly increased comparing with control (*: p<0.05) (F).</p

Ascl2 blockade in HT-29 and LS174T cells results in the inhibition of colony formation, proliferation, invasion and migration in vitro.

<p>shRNA-Ascl2/HT-29 and shRNA-Ascl2/LS174T cells have fewer colonies (*: p<0.05) (A), lower growth rates (*: p<0.05) (B), less invaded cells through the Matrigel-coated membrane (**: p<0.01) (C) and less cells migrating across the scraped edge (*: p<0.05) (D), when compared with their controls (Original magnification: ×200).</p

Knockdown of Ascl2 leads to the inhibition of the tumorsphere formation.

<p>1000 cells of shRNA-Ascl2/HT-29, shRNA-Ctr/HT-29 and non-transfected HT-29 cells are plated for tumorsphere formation as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032170#s2" target="_blank">Materials and methods</a> and quantified at 100× and 400× magnifications (A). The number of tumorspheres from shRNA-Ascl2/HT-29 cells is significantly less than that from shRNA-Ctr/HT-29 and non-transfected HT-29 cells (*: p<0.05) (B). The number of cells per tumorsphere from shRNA-Ascl2/HT-29 cells is significantly less than that from shRNA-Ctr/HT-29 cells and non-transfected HT-29 cells (*: p<0.05) (C).</p

The expression level of ‘stemness’ associated genes are reduced due to Ascl2 knockdown in vivo.

<p>The mRNA levels of CD133 (not performed in the tumor tissues developed from shRNA-Ascl2/LS174T and shRNA-Ctr/LS174T), Lgr5, Oct4, Bmi1, Sox2, and C-myc, analyzed by real-time PCR in the tumor tissues developed from shRNA-Ascl2/HT-29 and shRNA-Ascl2/LS174T cells are lower than in the tumor tissues developed from shRNA-Ctr/HT-29 and shRNA-Ctr/LS174T cells (*: p<0.05; **: p<0.01) (A). The protein levels of CD133 (not performed in the tumor tissues developed from shRNA-Ascl2/LS174T and shRNA-Ctr/LS174T), Oct4, Bmi1, Sox2, and C-myc, analyzed by western blot analysis in the tumor tissues developed from shRNA-Ascl2/HT-29 and shRNA-Ascl2/LS174T cells are significantly lower than in the tumor tissues developed from shRNA-Ctr/HT-29 and shRNA-Ctr/LS174T cells (B).</p

The percentage of CD133⁺ HT-29 cells and the expression level of ‘stemness’ associated genes are reduced due to Ascl2 knockdown in vitro.

<p>54.7% of shRNA-Ctr/HT-29 cells are positive for CD133 expression compared with 26.2% of HT-29 cells are positive for CD133 expression in shRNA-Ascl2/HT-29 cells (*: p<0.05) (A and B). The mRNA levels of ‘stemness’ associated genes, like CD133 (not performed in LS174T cells and its transfectants), Lgr5, Oct4, Bmi1, Sox2, and C-myc analyzed by real-time PCR in the shRNA-Ascl2/HT-29 and shRNA-Ascl2/LS174T cells are lower than in the shRNA-Ctr/HT-29, HT-29, shRNA-Ctr/LS174T and LS174T cells, respectively (*: p<0.05; **: p<0.01) (C). The protein levels of ‘stemness’ associated genes, like CD133 (not performed in LS174T cells and its transfectants), Oct4, Bmi1, Sox2, and C-myc, analyzed by western blot analysis in shRNA-Ascl2/HT-29 and shRNA-Ascl2/LS174T cells are significantly lower than that in the shRNA-Ctr/HT-29, HT-29, shRNA-Ctr/LS174T and LS174T cells, respectively (D).</p