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. 2012 Nov 14;288(1):141–151. doi: 10.1074/jbc.M112.431411

FIGURE 5.

FIGURE 5.

DKK1 regulates MT1-MMP-dependent collagen invasion. A, a modified Boyden chamber assay was used to evaluate invasion of cells through three-dimensional CI. Cells were transfected with control siRNA (siCtrl), DKK1-specific siRNA (siDKK1), or a DKK1 expression plasmid (pCS2+/DKK1; designated DKK1) or were incubated in the presence of 0.33 μg/ml recombinant DKK1 (rDKK1) as indicated. Cells invaded through the collagen gel (18 h) and attached to the lower surface of the filter. Following removal of non-invasive cells from the upper surface, the filter was stained, and invasive cells were quantified. Invasion of DOV13 cells was arbitrarily set as “1,” and other values were normalized to this value. An average of five independent experiments is presented. Error bars represent S.D. p values were obtained by Student's t test in comparison with non-treated DOV13 controls. * designates p < 0.0005. B and C, DOV13 cells were cultured on three-dimensional (3D) CI or planar (two-dimensional (2D)) collagen I in the presence or absence of 0.33 mg/ml exogenous DKK1 and subjected to Western blot to detect MT1-MMP expression (1:1000 antibody dilution). β-Tubulin was used as a protein loading control. The histogram (C) shows densitometric quantitation of MT1-MMP levels. Expression of MT1-MMP in cells on planar collagen I was arbitrarily set as 1. p values were obtained by Student's t test. * designates p < 0.005 relative to culture on three-dimensional CI. D, DOV13 cells were cultured on three-dimensional CI or planar (two-dimensional) collagen I in the presence or absence of 0.33 mg/ml exogenous DKK1 for 4 (open bars) or 8 h (solid bars). RNA was extracted, cDNA was synthesized, and real time RT-PCR was performed to detect MT1-MMP RNA expression. Ratios of MT1-MMP RNA expression were found using the 2−ΔΔCt method. p values were obtained by Student's t test. * designates p = 0.03 relative to the 4-h sample in the absence of DKK1, ** designates p = 0.002 relative to the 4-h sample in the absence of DKK1, and *** designates p = 0.001 relative to the 8-h sample in the absence of DKK1.