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. 2011 Sep 23;108(41):17135–17140. doi: 10.1073/pnas.1104182108

Fig. 3.

Fig. 3.

Colorectal cancer cell differentiation is induced by β-catenin shRNA in vivo. (A–D) LS411N cancer cells containing NTC, sh35, or sh36 β-catenin shRNA were inoculated into mice. Tumor-bearing mice were treated for 3 d with either vehicle or doxycycline. (A) Quantitative RT-qPCR of tumor expression of crypt progenitor markers including EPHB2, LGR5, and BMP4 and differentiation markers including MUC2, CA2, and TM4SF4 after 3 d of treatment. Expression was normalized to 18S mRNA. Graphs represent mean ± SEM values. Arbitrary units are shown. (B) Representative images of EPHB2 staining (Left) by IHC after 3 d of treatment. Percentages of positive pixels are indicated. Graphs represent mean ± SEM values. (Center) Representative images of Alcian blue staining after 3 d of treatment. Percentages of blue pixels are indicated. Graphs represent mean ± SEM values. (Right) Representative images of CA2 staining by IHC after 3 d of treatment. Percentages of positive pixels are indicated. Graphs represent mean ± SEM values. Two independent experiments are represented (n = 3 per treatment group). (C) Representative electron microscopy images of sh36 β-catenin shRNA tumors treated for 3 d with either vehicle (Upper) or doxycyline (Lower). Arrow indicates apical–lateral junction. (D, Left) Representative images of KLF4 (green) and p21 (red) costaining by immunofluorescence after 3 d of treatment. (Right) Mean intensity of nuclear KLF4 as a function of mean intensity of nuclear p21 in the indicated conditions. Each dot represents one nucleus. At least 1,000 nuclei were analyzed per condition. Correlation between mean intensity of nuclear KLF4 and mean intensity of nuclear p21: P < 0.0001 (Spearman's correlation test).