Fig. 6

A Control and RNF12 stably depleted MDA-MB-231 cells were plated for the cell wound-healing assay. Sub-confluent MDA-MB-231 cells were wounded using a 20 µL pipette tip and treated with or without the indicated ligands and reagents for 16 h. The wound-healing area at 16 h after wounding was normalized to that at the start time. B Statistical analyses were performed with Student’s t-test (*0.01 < p < 0.05; **0.001 < p < 0.01; ***p < 0.001). For the bar charts, data are plotted as the mean ± SD of at least three independent experiments. C Immunoblotting analysis of mesenchymal marker expression treated with IGF-1, TGF-β, and MK2206 as indicated. D RNF12 depletion inhibits AKT- and TGF-β-induced EMT in MDA-MB 231 cells. Cells were treated with TGF-β for 24 h. F-actin was stained with phalloidin conjugated with tetramethylrhodamine isothiocyanate (TRITC). E Representative images of RNF12 and matched pAKT and pSMAD2 immunohistochemistry results are shown. Graphs in the right panel show the correlation between RNF12 expression levels and those of pSMAD2 in breast cancer TMAs. Spearman’s test was used, and the correlation coefficients and the two-tailed significance values (p < 0.001) are shown. Scale bar = 300 μm. F Proposed model for the crosstalk between AKT signaling and RNF12 in breast cancer metastasis.