Fig. 4: IK KO induced changes in metabolism and Ca²⁺ elevate AMPK activity.

A Western blot analysis of GAPDH, AMPK, and phosphorylated AMPK at Thr¹⁷² in protein lysates obtained from MMTV-PyMT WT (left) and MMTV-PyMT IK KO (right) cells. Data represents average ±SEM of n = 6 independent experiments per genotype. B Quantification of Western blot band intensities as seen in A and normalization on GAPDH of MMTV-PyMT WT (black bar) and MMTV-PyMT IK KO (red bar), respectively. Bars represent average ±SEM, n = 6 with **p ≤ 0.01, unpaired t-test. C Demonstrates ratio of phosphorylated AMPK to whole AMPK intensities normalized on GAPDH of MMTV-PyMT WT (black bar) and MMTV-PyMT IK KO (red bar). Bars represent average ±SEM, n = 6 with ***p ≤ 0.001, unpaired t-test. D 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 AMPKAR, a FRET-based AMPK activity reporter (AMPK_act). Extracellular removal of Ca²⁺ (0 mM Ca²⁺ + EGTA), administration of Ionomycin, or Ca²⁺ (2.0 mM) re-addition were performed as indicated in the panel. Data represents average ±SEM of n = 5 independent experiments per genotype. Scalebar = 20 µm. E FRET-ratio signals over time of MMTV-PyMT WT (black line) and MMTV-PyMT IK KO cells (red line) expressing AMPKAR. Administration of extracellular ATP, i.e., triggering of intracellular Ca²⁺ release, reveals changes in AMPK activity at indicated timepoints. Data represents average ±SEM of n = 5 independent experiments per genotype. F Basal FRET-ratio values (timepoints 0–5) and G the difference of maxima- (I) to the endpoint (II) FRET-ratios of MMTV-PyMT WT (black bars) and MMTV-PyMT IK KO cells (red bars), of curves as shown in E. Bars represent average ±SEM, n = 5 with ***p ≤ 0.001, unpaired t-test. H FRET-ratio signals over time of MMTV-PyMT WT (black line) and MMTV-PyMT IK KO cells (red line) expressing AMPKAR. Administration of glycolysis inhibitor 2-DG reveals changes in AMPK activity at indicated timepoints. Data represents average ±SEM of n = 6 independent experiments per genotype. I Basal (timepoints 0–5) and J maximal FRET-ratio signals of MMTV-PyMT WT (black bars) and MMTV-PyMT IK KO cells (red bars) expressing AMPKAR. I, J demonstrate corresponding FRET-ratio signals of curves shown in H, either under basal conditions (timepoints 0–5 in (I)) or after treatment with 2-DG (J). Bars represent average ±SEM, n = 6 with ***p ≤ 0.001, unpaired t-test with Welch’s correction for different varriances. K FRET-ratio signals of MMTV-PyMT WT (black line) and MMTV-PyMT IK KO cells (red line) expressing AMPKAR over time. Administration of ATP synthase inhibitor Oligomycin-A reveals changes in AMPK activity at indicated timepoints. Data represents average ±SEM of n = 6 independent experiments per genotype. L Basal (timepoints 0–5) and M maximal FRET-ratio signals of AMPKAR expressing MMTV-PyMT WT (black bars) and MMTV-PyMT IK KO cells (red bars) of curves as demonstrated in K. Bars represent average ±SEM, n = 6 with **p ≤ 0.01, unpaired t-test. N Summarizing scheme of the influence of IK channel deficiency (red arrows) on energy yield and AMPK activation. Figure created using BioRender.