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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 7. — Analyses of the requirement of AP4 and SNAIL for EMT-associated traits. (A and B) Immunoblot (A) and qPCR analysis (B) of SW480 cells harboring episomal plasmids for conditional expression of two different miRNAs directed against AP4 (AP4mi1 or AP4mi2) or a nonsilencing miRNA (NonS). For generation of the miRNAs, see Materials and methods. Cells were treated with DOX for 48 h. (C) Transwell Boyden chamber assay of cellular migration or invasion after conditional down-regulation of AP4 in SW480 cells. (D and E) 96 h after AP4 activation by addition of DOX and concomitant SNAIL-specific siRNA transfection of DLD-1 cells, lysates were subjected to immunoblot analysis of the indicated proteins (D) or phase-contrast pictures were obtained (E). (F) Transwell Boyden chamber assay of cellular migration and invasion in DLD-1 cells with and without AP4 activation by addition of DOX and/or transfection of SNAIL-specific siRNAs for 96 h. For the last 24 h, the serum concentration was reduced to 0.1%. To analyze invasion, membranes were coated with Matrigel. After 48 h, cells were fixed and stained with DAPI. The mean number of cells in five fields per membrane was counted in three different inserts. Relative invasion or migration is expressed as the cell number in the test versus the control (ctrl. siRNA or without DOX) samples. (G and H) 96 h after SNAIL activation by DOX and concomitant AP4-specific siRNA transfection of DLD-1 cells, lysates were subjected to immunoblot analysis of the indicated proteins (G) or phase-contrast pictures were obtained (H). (E and H) Bars, 50 µm. (I) Transwell Boyden chamber assay as in F but after SNAIL activation and AP4 down-regulation as indicated. For siRNA transfections (D–I), identical total siRNA concentrations (20 nM) were achieved by reconstitution with control siRNA. Experiments in B, C, F, and I were performed in triplicates (n = 3); error bars indicate SD; significance level as indicated: *, P < 0.05; ***, P < 0.001.