Figure 5.

β1-integrin blocking antibody prevents nucleolar simplification (A-D) MCF10A cells were cultured in 2D (A) and 3D (B) in the presence or absence of anti-β1-integrin AIIB2 antibody and examined by phase contrast microscopy over 14 days. During this period, AIIB2 had no effect on cells grown in 2D culture, but it prevented the formation of acini in 3D culture, even though very low levels of apoptosis were seen. The disruption of acinus formation was evident when 3D cultures (C) were stained for laminB1 (green) and fibrillarin (red) and this disruption correlated with the loss of nucleolar simplification that was seen in control mature acini (D; CT; n = 176; AIIB2; 7-d n = 98 and 14-d n = 146). Nucleolar numbers in 3D cultures incubated with isotype IgG mimicked those seen in untreated controls whereas cultures treated with AIIB2 had nucleolar profiles similar to cells grown in 2D culture (see Fig. 1B). The maturation of acini treated with isotype control IgG correlated with increased expression of SA-β-gal (E, F), whereas no expression was seen in 2D cultures and much reduced expression was seen in cultures treated with AIIB2 (E, F), implying that the normal function of β1-integrin was necessary to maintain expression of the senescence marker. The proliferative capacity of cells in 3D culture declined (G) as judged by EdU incorporation, whether or not AIIB2 was present. However, mature acini formed under control conditions returned to cycle much more efficiently than AIIB2-treated acini on re-plating into planar cultures (H; images shown in Fig. S6). Scale bars, 10 μm.