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. 2013 Jul 1;210(7):1331–1350. doi: 10.1084/jem.20120812

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© 2013 Jackstadt et al.

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Figure 9. — Requirement of AP4 for metastasis in a xenograft mouse model. (A) Detection of c-MYC and AP4 protein in the indicated CRC cell lines by immunoblot analysis. (B and C) SW620 cells stably expressing luciferase were transfected with the indicated siRNAs. 72 h later, protein lysates were subjected to immunoblot analysis of the indicated proteins. (A–C) Detection of α-tubulin served as loading control. (D) Transwell Boyden chamber assay of cellular invasion. SW620-Luc cells were transfected with the indicated siRNAs. After 72 h, cells were seeded into a transwell chamber and analyzed as in Fig. 7 G. (E) SW620-Luc cells were transfected with the indicated siRNAs. After 72 h, cells were injected into the tail veins of NOD/SCID mice (n = 6 for each siRNA). 9 wk later, representative bioluminescence images were obtained. (F) Bioluminescence signals recorded at the indicated time points are represented as total flux. (G) Lungs were resected 9 wk after injection of SW620-Luc cells. Arrows indicate metastatic tumor nodules. (H) Detection of human Vimentin (red) in the lungs of mice injected with SW620-Luc cells transfected with the indicated siRNAs. Bars, 500 µm. (I) Quantification of metastatic nodules in the lung per mouse 9 wk after injection of SW620-Luc cells transfected with the indicated siRNAs. For the analysis in D (n = 3) and F and I (n = 6), error bars represent SD; significance level as indicated: **, P < 0.01; ***, P < 0.001.