Fig. 3: Loss of IK impacts subcellular Ca²⁺ homeostasis.

A Representative FRET (left panel, yellow, left), CFP (left panel, cyan, middle), and pseudocoloured FRET-ratio images (left panel, 16-colors, right) and FRET-ratio signals over-time (right panel) of MMTV-PyMT WT (black) and MMTV-PyMT IK KO cells (red) expressing 4mtD3cpv, a FRET-based mitochondrial Ca²⁺ indicator ([Ca²⁺]_mito). At time point indicated in the panel, either extracellular ATP, to trigger release of Ca²⁺ from intracellular stores via metabotropic receptors, or Oligomycin-A for inhibition of ATP synthase were administered. Scalebar = 20 µm. B Area under the curve (AUC) from timepoints 5–15 min of FRET-ratio signals as shown in A. Data represents average ±SEM, n = 6 with ***p ≤ 0.001, unpaired t-test with Welch’s correction for different varriances. C Fluorescence emission ratio signals of MMTV-PyMT WT (black line) and MMTV-PyMT IK KO cells (red line) loaded with FURA-2 over-time in response to administration of extracellular ATP at time point indicated in the panel. Data represents average ±SEM, n = 6 independent experiments per genotype. D Area under the curve (AUC) from timepoints 5–12.5 min of FRET-ratio signals as shown in C represent average ±SEM, n = 6 with ***p ≤ 0.001, unpaired t-test with Welch’s correction for different varriances. E Representative images of MMTV-PyMT WT (left panel, upper lane, and right panel, black curve) and MMTV-PyMT IK KO cells (left panel, lower lane, and right panel red curve) loaded with FURA-2, either at an excitation of 340 nm (left panel, pink, left) or 380 nm (left panel, violette, middle). Right images show pseudocoloured ratio image (left panel, 16-colors, right). FURA-2-ratio signals were recorded over-time (right panel) of MMTV-PyMT WT (black) and MMTV-PyMT IK KO cells (red) in response to extracellular removal of Ca²⁺ (0 mM Ca²⁺ + EGTA), administration of BHQ for SERCA inhibition, or cell stimulation with ATP at indicated timepoints. Data represents average ±SEM of n = 6 independent experiments per genotype. Scalebar = 20 µm. In F basal FURA-2 ratio signals (timepoints 0–5 in E), G the difference of basal- to the minimal FURA-2 ratios of timepoints 5–10 min and H area under the curve (AUC) from timepoints 10–20 min of MMTV-PyMT WT (black bars) and MMTV-PyMT IK KO cells (red bars) are shown. Data represent means ± SEM of n = 6 with ***p ≤ 0.001, unpaired t-test with Welch’s correction for different varriances. I Normalized fluorescence over time signals of MMTV-PyMT WT (black line) and MMTV-PyMT IK KO cells (red line) expressing Car-GECO1, a single FP-based, red fluorescent Ca²⁺ sensor located in the cytosol ([Ca²⁺]_cyto). At time points indicated in the panel, extracellular Ca²⁺ was removed (0 mM Ca²⁺ + EGTA), Ionomycin was added and extracellular Ca²⁺ (2.0 mM) was re-added. Data represents average ±SEM of n = 5 independent experiments per genotype. J Basal Ca²⁺ MMTV-PyMT WT (black bar) and MMTV-PyMT IK KO cells (red bar) expressing Car-GECO1 (timepoints 0–5 in I). Bars represent average ±SEM, n = 5 with *p ≤ 0.05, unpaired t-test. K Basal FRET-ratio signals (timepoints 0–5) of MMTV-PyMT WT (black bar) and MMTV-PyMT IK KO cells (red bar) expressing D1ER ([Ca²⁺]_ER), a FRET-based Ca²⁺ indicator targeted to the endoplasmic reticulum (ER). Data represents average ±SEM of n = 3 with ***p ≤ 0.001, unpaired t-test. L Putative consequences of IK channel deficiency on Ca²⁺ signaling pathways in MMTV-PyMT BC cells. Figure created using BioRender.