Figure 4.

Soft substrate disturbs Ca²⁺ homeostasis. (A) Soft substrate up-regulates Ca²⁺ influx in normal cervical epithelial cells. Cells were cultured on control condition or on soft substrate for 0.5, 1, and 4 h. To measure Ca²⁺ entry across the plasma membrane, normal cervical epithelial cells were preincubated in Ca²⁺-free media plus 2 μM thapsigargin (TG) for 30 min to deplete the internal Ca²⁺ storage. Each trace is mean ± SEM of at least 30 single cells. (B) Basal [Ca²⁺]_i was calculated by Fura-2/AM measurement. Before [Ca²⁺]_i measurement, normal cervical epithelial cells were cultured on the control condition or soft substrate for 4 h in the presence of 2 mM [Ca²⁺]_o. Each column is mean ± SEM from at least 100 cells. (C) Cytosolic Ca²⁺ levels were estimated by Fluo-4/AM measurement. Representative pictures of phase contrast, green fluorescent intensity and pseudocolor Fluo-4 image in various culture conditions. Before measurement, normal cervical epithelial cells were cultured on the control condition or soft substrate for 4 h in the presence of 2 mM [Ca²⁺]_o. Scale bar, 20 μm. (D) Quantification of Fluo-4 fluorescent intensity. Each column represents mean ± SEM from at least 100 different cells. (E) Model of FRET-based Ca²⁺ measurement by cameleon. M13, CaM-binding domain of myosin light-chain kinase. (F) Top panel, pseudocolor images indicating emission ratio of EYFP to ECFP for normal cervical epithelial cells seeding on soft substrate or control condition for 4 h in the presence of 2 mM [Ca²⁺]_o. Scale bar, 10 μm. Bottom panel, quantification of emission ratio of EYFP to ECFP. Each column is mean ± SEM from at least 30 cells.