Figure 3. A thick glycocalyx stimulates integrin-FAK mechanosignaling.
(A) Immunoblot analysis of FAK phosphorylation 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. (B) Western blots from A were analyzed by densitometry, their values normalized first to total FAK then to that of the positive control—PBS treated cells on a stiff fibronectin-functionalized matrix. (C) Immunoblot analysis and quantification of pFAK and total FAK in 4TO7 cells treated as in A and quantified as in B except normalized to short glycopolymer control. D) pFAKY397 IF staining in green of mets from experiments in Figure 1. DAPI nuclear stain in blue. (E) Quantification of IF staining in D normalized to the average signal per nuceli in mets from short glycopolymer-treated cells. Shown is mean ±SEM of three mice per group from which 3–4 tumors were averaged each. (F) A model by which a mucin-bolstered glycocalyx may drive proliferation. Limited ligand access due to steric hindrance establishes a kinetic funnel in which integrins are likely to bind where bonds exist already. This drives a clustering of integrins that activates FAK which, in conjunction with EGFR, drives activation of Akt and subsequently cell cycle progression. Blot in A is representative of at least three biological replicate experiments. Shown in B is mean ±SEM from five biological replicate experiments. Shown in C is mean ±SEM from three biological replicate experiments. Scale bars are 100 μm. *p<0.05 (Student’s paired t-test).
Figure 3—figure supplement 1. FAK inhibitor abolishes proliferative advantage from bulky glycocalyx.
