Figure 5.

Overexpression of TRPC6 attenuates melatonin-evoked inhibition of store-operated Ca²⁺entry in MDA-MB-231 cells.A, left panel, MDA-MB-231 cells were transfected with TRPC6dn-YFP or TRPC6-YFP expression plasmids. Forty-eight hours later, TRPC6 expression was determined by fluorescence microscopy. A, right panel, MDA-MB-231 cells were transfected with TRPC6dn-YFP or TRPC6-YFP expression plasmids or empty vector (control). Forty-eight hours later cells were loaded with fura-2. Fura-2-loaded cells were perfused with a Ca²⁺-free medium (100 μM EGTA added) and then stimulated with TG (1 μM) followed by reintroduction of external Ca²⁺ (final concentration 1 mM) to initiate Ca²⁺ entry. Scatter plots represent the quantification of TG-evoked Ca²⁺ release and entry determined as described in Experimental procedures. Data are presented as mean ± SD. Dots represent a single experiment including 20–30 cells. Analysis of statistical significance was performed using one-way ANOVA (F = 1.82; p = 0.19 for Ca²⁺ release and F = 546.5 and p < 0.0001 for Ca²⁺ entry, with post-hoc Dunnett's test; ∗p < 0.0001 compared with control). B–E, MDA-MB-231 cells were treated with melatonin (10–1000 nM) or the vehicle for 72 and 168 h. Forty-eight hours before the end of the treatment period, cells were transfected with TRPC6-YFP expression plasmid. Fura-2-loaded cells were perfused with a Ca²⁺-free medium (100 μM EGTA added) and then stimulated with TG (1 μM) followed by reintroduction of external Ca²⁺ (final concentration 1 mM) to initiate Ca²⁺ entry. D and E, quantification of TG-evoked Ca²⁺ release and entry. Scatter plots are represented as mean ± SD. Dots represent single experiments including 20–30 cells. Data obtained from cells transfected with TRPC6-YFP are compared with those obtained in control cells (gray bars; presented in Fig. 3) performed in parallel. Analysis of statistical significance was performed using two-way ANOVA (F values were 0.28, 0.59, and 0.32 and p values were 0.83, 0.61, and 0.96 for concentration, time, and the interaction respectively for Ca²⁺ release; F values were 769.6, 2479, and 80.87 and p values were <0.0001 in all cases for Ca²⁺ entry) with post-hoc Tukey test (∗p < 0.0001 compared with the response observed in vehicle-treated cells). F–G, MDA-MB-231 cells were treated with melatonin (10–1000 nM) or the vehicle for 72 and 168 h. Forty-eight hours before the end of the treatment period, cells were transfected with TRPC6dn-YFP expression plasmid or empty vector. Cells were loaded with propidium iodide, and cell staining was visualized using an inverted microscope as described in Experimental procedures. Images shown are representative of three independent experiments.