Figure 7.

Effect of pharmacological inhibition or silencing of TRPC6 on Ca²⁺entry in the presence of melatonin in MDA-MB-231 cells.A, Fura-2-loaded MDA-MB-231 cells were pretreated for 10 min with SAR7334 (1 μM). Cells were then stimulated in the presence of 1 mM extracellular Ca²⁺ with OAG (100 μM). Scatter plots represent the quantification of OAG-induced Ca²⁺ 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 Student's t-test (∗p < 0.0001 as compared with control). B–D, MDA-MB-231 cells were treated for 72 h with melatonin (100–1000 nM) or the vehicle (control). The day of the experiment, fura-2-loaded cells were pretreated for 10 min with SAR7334, 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. Data are presented as mean ± SD. Dots represent a single experiment including 20–30 cells. Analysis of statistical significance was performed using Student's t-test (∗p < 0.0001 as compared with control). E, MDA-MB-231 cells were treated with melatonin (100–1000 nM) or the vehicle (control) for 72 h. Forty-eight hours before the end of the treatment period, cells were transfected with shTRPC6. 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 = 0.74/p = 0.47 and F = 0.63/p = 0.53 for Ca²⁺ release and entry, respectively; with post-hoc Dunnett's test for comparison between the data corresponding to Ca²⁺ entry [∗p < 0.0001 as compared with control]). F, MDA-MB-231 cells were treated with melatonin (100–1000 nM) or the vehicle (control) for 72 h. Forty-eight hours before the end of the treatment period, cells were cotransfected with Orai1 and OASF. The day of the experiment, fura-2-loaded cells were perfused with a Ca²⁺-free medium (100 μM EGTA added), followed by reintroduction of external Ca²⁺ (final concentration 1 mM) to initiate Ca²⁺ entry. Scatter plots represent the quantification of Ca²⁺ entry. 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 = 0.21; p = 0.80).