Fig. 3

Downregulation of Irf6 is required for ErbB2-induced anoikis resistance of breast epithelial cells. a MCF-ErbB2 cells were transiently transfected or not with a control vector (pcDNA3) or pcDNA3 vector encoding hemagglutinin (HA)-tagged Irf6 (pcDNA-HA-Irf6) and a vector encoding green fluorescent protein (GFP; vector pEGFP-C1) and assayed 24 h later for HA-Irf6 expression by Western blotting using an anti-HA antibody. b Cells treated as in (a) were cultured detached from the extracellular matrix (in 3D culture) for 72 h, cell nuclei were stained with Hoechst 33258, and the percentage of GFP-positive cells with condensed and/or fragmented nuclei (characteristic features of apoptosis) was determined as the percentage of the cells with such nuclei in a population of GFP-positive cells. c MCF-ErbB2 cells infected with the control or the Irf6-encoding Moloney murine leukemia virus were kept in 3D culture for 3 h along with MCF10A cells and assayed for Irf6 levels by Western blotting. d MCF-ErbB2 cells treated as in (c) were kept in 3D culture for 24 h, stained with propidium iodide (PI), and assayed for annexin V binding by flow cytometry. % Apoptosis is the sum of the percentage of annexin V-positive/PI-negative cells and that of annexin V-positive/PI-positive cells. e MCF-ErbB2 cells treated as in (c) were allowed to form colonies in soft agar. The number of colonies formed by one of the replicates of the control cells was designated as 100%. The data in (b, d) are the average of two independent experiments plus the SD, and those in (e) are the average of three independent experiments plus the SE. β-actin was used as a loading control in (a, c). * p < 0.05