Fig. 4. Changes in [Ca²⁺]_i after hypotonic treatment. HaCaT and CHO cells were loaded with the Ca²⁺ indicator Fluo-4. Peak fluorescence intensities within 90 seconds after hypotonic treatment are plotted as ratios to control conditions (t0 seconds). (A) HaCaT cells exposed to hypotonic (hypo) conditions showed a significant increase in [Ca²⁺]_i compared with isotonic (iso) conditions (P<0.01, ANOVA). By contrast, hypotonically treated cells that were preincubated with 100 µM Gd³⁺ or kept in Ca²⁺-free solution showed no significant increase in [Ca²⁺]_i. Data are means±s.e.m. (isotonic, n=12; hypotonic, n=18; Gd³⁺, n=8; Ca²⁺-free, n=8). (B) EGFP-TRPV4-transfected CHO cells (TRPV4) exposed to hypotonic conditions showed a significant increase in [Ca²⁺]_i compared with untransfected cells (nt, P<0.01, ANOVA). This increase in [Ca²⁺]_i was absent from transfected cells pretreated with Gd³⁺ or kept in Ca²⁺-free solution. Data are means±s.e.m. (untransfected, n=22; EGFP-TRPV4, n=16; Gd³⁺, n=14; Ca²⁺-free, n=8).