Figure 2.

3D culture induces prolonged cell cycle arrest and expression of senescence markers in breast epithelia (A-E) MCF10A proliferation in 2D and 3D culture was assessed by EdU incorporation (green) and nucleoli labeled with anti-fibrillarin (red). Quantification of EdU-positive cells (2D: n = 210 (at 7d of culture); 3D: 7-d n = 186, 14-d n = 179, 21-d n = 240) showed a gradual decline in proliferation in 3D culture (B; S phase index was 23% at 1-d and 5–7% by 7-d). As cells exited cell cycle, expression of the cell cycle regulators cyclin D1 and p-RB declined (immunoblots shown in C) and the senescence markers p21 (C) and SA-β-gal increased (D, E); (D) shows typical phase contrast images on acini stained for SA-β-gal expression and (E) quantification of the stain (n = 25 acini). (F-I) Primary murine MECs grown in 3D (F) and 2D (H) cultures were stained for SA-β-gal activity. Note that no SA-β-gal was detected in 2D cultures whereas expression was clearly seen after 7 days in 3D culture. Changes in cell proliferation in these cultures was measured by EdU incorporation (30 min) after the indicated number of days in 3D (G) and 2D (I) culture. Scale bars, 50 μm.