Fig. 1

ErbB2 downregulates Irf6 in detached breast epithelial cells. a MCF10A and MCF-ErbB2 cells were cultured attached to (2D culture) or detached from (3D culture) the extracellular matrix for the indicated times and assayed for ErbB2 levels by Western blotting. b MCF10A and MCF-ErbB2 cells were cultured in 3D culture for 2 h, and Irf6 messenger RNA (mRNA) levels were analyzed in the cells by qPCR. Irf6 mRNA levels were normalized to those of 18S ribosomal RNA (determined by qPCR). The resulting Irf6 mRNA levels in one of the replicates derived from MCF10A cells were designated as 100%. Results represent the average of two independent experiments plus the SD. * p < 0.05. c MCF10A and MCF-ErbB2 cells were kept as in (b) for 3 h and assayed for Irf6 levels by Western blotting. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as a loading control. d MCF10A and MCF-ErbB2, as well as human breast carcinoma cells BT-474, AU-565, and HCC-1419, were kept in 3D culture for 2 h and assayed for ErbB2 levels by Western blotting. e Indicated cells were cultured in 3D culture for 48 h in the absence or presence of 1 μM lapatinib and assayed for Irf6 expression by Western blotting. f BT-474 cells or their trastuzumab-resistant variant were kept in 3D culture for 48 h in the absence or presence of 5 μg/ml trastuzumab and assayed for Irf6 expression by Western blotting. β-actin was used as a loading control in a and d–f, and GAPDH served as a loading control in c