Figure 2

LOXL2 expression disrupts normal breast epithelial acini formation. 10A cont and 10A L2 cells were plated on top of a thin layer of 50 μl Matrigel in eight-well chamber slides in a suspension of Matrigel/culture media mix to investigate the acinar morphogenesis of these cells. The cultures were allowed to grow for 8, 10, and 13 days, and then fixed and stained with primary antibodies, as described in the different panels. All data were based on at least three independent experimental repeats. Scale bar, 20 μm. (A) Immunofluorescence staining of acini with anti-activated caspase-3 antibody to detect apoptotic cells on day 8 reveals decreased apoptosis in the central cells of the L2 acini. Representative images of activated caspase-3 staining in acini for each cell line are shown. Quantification represented average percentage ± standard error of acini containing activated caspase-3-positive cells (P = 0.0007). (B) Immunofluorescence staining of acini with anti-Ki67 antibody to detect proliferating cells on day 13 reveals that L2 acini had more proliferative cells. Representative images of Ki67-positive acini from each cell line are shown. Quantification represented average percentage ± standard error of acini containing Ki67-positive cells. (P = 0.0007). (C) Immunofluorescence staining of acini with anti-GM130 antibody to assess cell polarity on day 10. Representative images of GM130 staining in acini for each cell line are shown. Quantification represented average percentage ± standard error of acini forming a regular ring structure, as assessed by GM130 staining (P = 0.0005; n = 80 acini for each cell line per repeat). (D) Quantification of average percentage of acini at day 13 with evacuated lumens ± standard error. Acini with evacuated lumens were defined as having no more than 20% of total number of cells, as well as Ki67-positive cells present in the center (P = 0.013).