Fig 1: TNBC cells display frequent IP3-linked Ca2+ oscillations. (A) Representative traces of cytosolic Ca2+ measurements (expressed as FURA-2 340/380 nm ratio, R) in MCF10A, MDA-MB-231 CTRL and MDA-MB-231 MCU-KO cells upon acute stimulation (arrows) by FCS (2%) or ATP (100 µM). Bars represent the corresponding quantification of ΔR upon FCS or ATP stimulation, as indicated. n = 74–88 cells from 3 independent experiments. (B) Representative traces of mitochondrial Ca2+ dynamics (expressed as mt-GCAMP6f 475/410 nm R) in MDA-MB-231 CTRL and MDA-MB-231 MCU-KO cells upon acute stimulation (arrow) by FCS (2%). Bars represent the corresponding quantification of ΔR. n = 22–45 cells from 3 independent experiments. (C) Representative traces of cytosolic Ca2+ dynamics (measured as in (A)) in MCF10A, MDA-MB-231 CTRL and MDA-MB-231 MCU-KO cells, bathed in a solution containing FCS (2%). Each trace represents a cell. On the right, bars represent the percentage of cells displaying cytosolic Ca2+ oscillations within 10 min (see Methods). Mean ± SEM. n = 92–153 cells from 3 independent experiments. (D) Representative western blots (WB) and quantification of IP3R-1 and IP3R-3 expression levels (normalized to actin) in the indicated cell types. Mean +SEM. n = 4-7 independent experiments. (E) Representative image of a MDA-MB-231 CTRL cell expressing GFP-PHD, before and after ATP stimulation. On the right, traces represent the fluorescence intensity (F) along the linear region of interest (yellow ROI) in the images on the left, to visualize changes in signal distribution before and after ATP stimulation. Scale bar, 10 μm. (F) Representative traces (left) and quantification (right) of the fluorescence change (ΔF/F0) in the cytosol of the indicated cell types expressing GFP-PHD, upon acute stimulation with FCS (2%, arrow). Mean ± SEM. n = 21–30 cells from 3 independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001.