Immunofluorescence (IF) staining was performed on paraffin-embedded tissue microarray (TMA) sections (Stanford TA390). Tissues were from patients with ductal carcinoma in situ. An International Review Board waiver was obtained for these studies, as the specimens were excess tissue not collected for the current research and were de-identified before use. The TMAs were cut into 4-μm-thick sections. Four-micrometer TMA sections were deparaffinized in three changes of xylene for 10 min each, hydrated in gradient series of ethyl alcohol, subjected to antigen retrieval, and then stained with p27Kip1 (1:2400 dilution) and α-tubulin (1:8000 dilution) for 45 mins at room temperature and counterstained with DAPI. IF stains were imaged using Ariol 3.4v (Leica Biosystems) at 40×. Five different fields of view of each human patient sample were randomly selected, and the Cell Profiler was used to quantify p27Kip1 localization and cell size. Note that images of 2D slices may not be directly comparable with 3D in vitro images, as the 2D slices may not capture the maximum cross section of any given cell and therefore underestimate the actual cell size. Because it was difficult to distinguish cell-cell boundaries in a human patient sample and to measure single-cell size, averaged measurements were performed instead of single-cell measurements. The averaged ratio of nuclear p27Kip1 to cytoplasmic p27Kip1 was calculated as the total intensity of nuclear p27Kip1 measured in 21 to 203 cells divided by the total intensity of cytoplasmic p27Kip1. The averaged cell size was calculated as the total area of cells divided by the total number of cells.

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