Fig. 4

Calcium dynamics in MDA-MB231 cells treated with L- and R-extracts. A Mean of intracellular Fluo-3 fluorescence measured day-by-day in a Nikon TE300 Inverted Fluorescence Phase Contrast Microscope, in L, R and Control conditions. Still Images of cohorts of 15–75 cells per condition were analyzed with Microsoft Excel and Image J (National Institutes of Health, USA, RRID:SCR_003070) during 5 days. Fluorescence data was calculated as mean/area. As can be observed, in the R-treated cells intracellular Ca²⁺ concentration decreases at day 1, (as compared to controls and to L-treated cells) and this difference is maintained along the 5 days (Kruskal–Wallis test for: L/R differences: p < 0.0001;Control/R differences: p < 0.001, indicated by asterisks). While L-treated cells, despite some fluctuations during days 2–3–4, do not significantly change from the untreated cells (Kruskal–Wallis test: Control/L differences: day 1, 2, 3 and 5: ns, day 4 p < 0.01). B The Δ fluorescence of A (initial fluorescence − final fluorescence) was measured at time 0 and 1440 min (24 h). This shows how R-treated cells decreased the intracellular Ca²⁺ concentration, which is not observed in the other conditions. C Representative Ca²⁺ fluorescence plot in real time, of single cells in L- and R-conditions, during 3 days. D Representative Ca²⁺ fluorescence plot in real time and representative single cell spatiotemporal fluorescence changes in the first 1440 min (24 h). Representative pictures of different times are shown (0, 60 and 550 min). The pseudocolor from black to red represents low to high concentration, respectively. E Scatter plot of real-time fluorescence in L- and R-conditions shown in C. F Plots of slopes of Ca²⁺ increments during the first 360 min (6 h) shown in C. The L-slope is markedly greater than R-slope