Fig. 4. Dormancy-associated fibronectin is assembled via α₅β₁ integrin and ROCK to mediate survival.

(A) Experimental timeline of inhibitor dosing. Day 7 experiments were dosed continually through a day 7 endpoint; day 28 experiments were dosed continually through a day 28 endpoint; separate cultures were established for 21 days, where inhibitor dosing was initiated for an additional 7 days (“dose-established culture”). (B) Day 7 survival of HCC 1954 on collagen with inhibitors dosed at seeding and every medium change. (C) Survival of cells with inhibitors, dosed after establishment of dormant culture for 21 days and then subsequent dosing at every medium change through days 21 to 28. (D) Day 7 survival of HCC 1954 on decellularized matrix with inhibitors dosed at every medium change. (E) Day 28 survival of cells with inhibitors dosed at seeding and every medium change throughout the entirety of the experiment. Green, α₅ integrin function affecting antibody; blue, Y-27632 (ROCK inhibitor at various concentrations). (F) Immunofluorescence for fibronectin (green) and DAPI (blue) at day 28 on collagen with inhibitors dosed for the entire 28-day time period. Scale bar, 100 μm. (G) Immunofluorescence for fibronectin (green) and DAPI (blue) at day 28 on collagen with exogenous fibronectin (10 μg/ml) or fibronectin with Y-27632 (1 μM). (H) Day 28 survival of cells under control, exogenous fibronectin, or fibronectin with Y-27632. P < 0.05 is denoted with *, P ≤ 0.001 with *** and P ≤ 0.0001 with ****.