Fig. 2

Detachment-induced upregulation of Irf6 is required for anoikis of nonmalignant breast epithelial cells. a, b MCF10A cells (a) or human mammary epithelial cells (HMEC) (b) were kept attached to (2D culture) or detached from (3D culture) the extracellular matrix for the indicated times and assayed for Irf6 levels by Western blotting. c MCF10A cells were kept in 2D or 3D culture for the indicated times and assayed for Blnk levels by Western blotting. d, e MCF10A cells transfected with 100 nM control RNA or Irf6-specific small interfering RNA 5 or 7 were detached for 3 h and assayed for Irf6 (d) or Blnk (e) expression by Western blotting. β-actin was used as a loading control in a–e. f MCF10A cells treated as in (d) were allowed to form colonies in monolayers immediately or after 72 h of detachment. % survival is the percentage of colony number formed by the cells plated in monolayers immediately after transfection. The data represent the average of three independent experiments plus the SE. g MCF10A cells treated as in (c) were detached for 48 h, then cell nuclei were stained with Hoechst 33258, and the percentage of cells with condensed and/or fragmented nuclei (characteristic features of apoptosis) in the total cell population (% apoptosis) was determined. The data represent the average of two independent experiments plus the SD. * p < 0.05