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

FIGURE 2.

FIGURE 2.

Three-dimensional collagen culture down-regulates DKK1 expression. A, DOV13 cells were cultured on three-dimensional CI (3D) or planar collagen (two-dimensional (2D)) for the indicated time followed by real time RT-PCR analysis of DKK1 mRNA expression. Shown is the -fold change in the ratio of DKK1 mRNA in three-dimensional relative to two-dimensional collagen. Ratios of DKK1 RNA expression were obtained using the 2−ΔΔCt method. An average of three independent experiments ±S.D. (error bars) is presented, and data were statistically evaluated by t test. * designates p < 0.05. B, multiple ovarian carcinoma cell lines as indicated were cultured on three-dimensional CI or planar collagen (two-dimensional) for 8 h followed by RNA extraction, cDNA synthesis, and real time RT-PCR to assess DKK1 mRNA levels. Shown is the -fold change in the ratio of DKK1 mRNA in three-dimensional CI relative to two-dimensional. * designates p < 0.05. C, DOV13 cells were cultured for 8 h on three-dimensional CI or planar collagen (two-dimensional) in the presence of DMSO, SU6656 (2 μm), or UO126 (25 μm) as indicated followed by analysis of DKK1 mRNA expression as in A. * designates p < 0.05. D, representative Western blot of DKK1 levels in whole cell lysates after 24-h culture on planar collagen (two-dimensional) and three-dimensional CI as indicated. Blots were developed using polyclonal anti-DKK1 (1:200) and anti-rabbit HRP-conjugated secondary antibodies (1:1000). Expression of β-tubulin was probed as a loading control. E, DOV13 cells were cultured atop three-dimensional RGDS-PEG gels or planar unconjugated RGDS for the indicated periods of time followed by RNA extraction, cDNA synthesis, and real time RT-PCR. Averaged ratios of DKK1 mRNA levels in cells cultured on three-dimensional versus two-dimensional substratum are plotted on the histogram as -fold down-regulation against time. * designates p ≤ 0.005. F, expression of DKK1 mRNA was analyzed in DOV13 cells cultured on three-dimensional gels prepared from type III collagen relative to cells cultured on planar (two-dimensional) collagen III. Additionally, expression was evaluated in cells subjected to mechanical strain (as described under “Experimental Procedures”) compared with control cells as indicated. Averaged ratios of DKK1 mRNA levels in cells cultured on three-dimensional CIII versus planar CIII (two-dimensional) and under conditions of strain versus controls are plotted on the histogram as -fold down-regulation. * designates p < 0.005.