Fig. 3: Aberrant ZEB2 expression induces EMT, migration and invasion of CRC cells in vitro and in vivo.
a WB analysis of DLD1 cells ± FBXW7 (left) and murine fbxw7fl/fl vs. fbxw7ΔG derived crypts and IMF proteins (right) using α-SMA, ZEB2, Vimentin, N-cadherin, E-cadherin antibodies, and β-actin loading control. b Top, ZEB2 IHC on the intestine from 3-week fbxw7fl/fl and fbxw7ΔG mice. Dashed lines indicate the boundary of the IMF and Ep. Red arrowheads show Ep and green arrowheads show IMF with different Zeb2 protein levels in fbxw7fl/fl vs. fbxw7ΔG. b Bottom, IHC for ZEB2 in samples of CRC patients with (n = 10) and without (n = 11) FBXW7 mutations. A boxed line indicates a magnified tissue area. Red arrowheads show Ep and green arrowheads show stromal cells with different ZEB2 protein levels. Scale bars, 50 μm. c Left, HCT116FBXW7(−/−) and HCT116FBXW7(+/+) cells with ZEB2 knockdown (ZEB2-shRNA) and scrambled vector (sc-shRNA) controls, stained with rhodamine–phalloidin marking F-actin filaments. Scale bars, 100 µm. c Right, WB analysis of HCT116 cells ± FBXW7, expressing the sc-shRNA controls and ZEB2-shRNA using ZEB2, Vimentin and E-cadherin antibodies. d Representative images of xenograft metastatic models containing disseminated sc-shRNA:FBXW7(+/+), sc-shRNA:FBXW7(−/−) and ZEB2-shRNA:FBXW7(−/−) HCT116 cells in the murine liver and lung. Tissues were stained with antibodies against human keratin5 (KRT5) (top panels) or against the cell tag GFP (bottom panels). Scale bars, 50 µm. e–h Total number of foci of disseminated cells or foci with size ≥40 µm of sc-shRNA:FBXW7(+/+), sc-shRNA:FBXW7(−/−) and ZEB2-shRNA:FBXW7(−/−) HCT116 cells in the liver (e, f) and lung (g, h) were manually counted in five views of KRT5 stained sections/mouse and per each cell line. Absolute number was normalised to control sc-shRNA:FBXW7(+/+) cell line. Bars represent mean ± SD, n = 5; *P < 0.05, **P < 0.01, ***P < 0.001, using Student’s t test
