(A) and (B) Microscopy of MCF-10A cells labeled with short or long glycopolymers, plated on soft (400 Pa) fibronectin-functionalized gels, and cultured for 72 hr. (C) Average number of cells per colony in images such as A and B. (D) Percent of colonies containing more than 2 cells in images such as A and B. (E) Immunoblot analysis of proliferative markers in MCF-10A cells coated with long or short glycopolymers or treated with vehicle (PBS) and plated on soft (400 Pa) or stiff (60 kPa) fibronectin-functionalized polyacrylamide gels for six hours. (F) Western blots from E were analyzed by densitometry, their values normalized first to total protein (tubulin) then to that of the positive control—PBS treated cells on a stiff Fn-functionalized matrix. (G) Immunoblot analysis of pAkt and pErk in MCF-10A cells serum-starved for 72 hr, treated with long or short glycopolymers or vehicle and plated in serum-free media on soft or stiff gels for six hours, then challenged with epidermal growth factor for 15 min, and lysed. (H) Western blots from G were quantified and normalized to total Akt then to the positive control. (I) Immunoblot and quantification of pAkt and total Akt in 4TO7 cells in vitro treated as in G and quantified as in H except normalized to short glycopolymer control. (J) Cyclin D1 (green) IF staining of 4TO7 mets from experiments in Figure 1. DAPI nuclear stain in blue. (K) Quantification of IF staining normalized to the average signal per nuceli in mets from short glycopolymer treated cells. Shown in K is the mean ±SEM of three mice per group from which 3–4 tumors were averaged each. For C, D, F, and I, shown is the mean ±SEM of three biological replicate experiments. For H, shown is the mean ±SEM of four biological replicate experiments. Scale bars are 100 μm. *p<0.05, **p<0.01 (Student’s paired t-test).