Fig. 4.

Phosphorylation of RNF41 by Par-1b is required for laminin-111 deposition and polarized localization of laminin-111 receptors. (A–C) MCF10A cells (control, knockdown and rescue cells, as described in Fig. 3C) were cultured in a collagen I matrix and on day 20, colonies were stained with antibodies against laminin-111 (A), βDG (B), or β1 integrin (C). Colonies were visualized with secondary antibody conjugated to Alexa Fluor 488 (green) and To-Pro-3 to detect nuclei (blue). A serial immunofluorescent confocal cross section of a representative colony is shown. Scale bars: 20 µm. The data presented is representative of the 500 colonies that were observed in each of three independent experiments, for a total of 1500 colonies. (D) Percentage of normal spheroids. The percentage of MCF10A cells in panels A–C that formed normal acini-like spheroids was determined. 500 colonies were counted in each of three independent experiments for a total of 1500 colonies. The data are presented as the mean (±s.e.m.) percentage of normal spheroids. ***P<0.001 (one-way ANOVA with a Tukey's multiple comparison post test); NS, not significantly different. (E,F) Lysates from 3D colonies were resolved by SDS-PAGE and analyzed by western blotting with antibodies specific for laminin-111, βDG, utrophin, β1 integrin or GAPDH. In addition, laminin-111 was immunoprecipitated from the culture medium and immunoprecipitates were analyzed by western blotting (IP, panel E). A representative image from n = 3 is shown.