Fig. 5: AMPK activation elevates autophagy in IK-deficient cells.
From: IKCa channels control breast cancer metabolism including AMPK-driven autophagy
A Western blot analysis of αTubulin, LC3B (I + II), ULK1, phosphorylated ULK1 at Ser555, p62 and GAPDH of 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 demonstrated in A and normalization of LC3B (I + II) on αTubulin and ULK1, pULK1 and p62 on GAPDH of MMTV-PyMT WT (black bar) and MMTV-PyMT IK KO (red bar). Bars represent average ±SEM, n = 6 with *p ≤ 0.05 and ***p ≤ 0.001, unpaired t-test. C Ratio of phosphorylated ULK1 to total ULK1 protein 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 immunofluorescence (IF) staining of DAPI (left panel, blue, left), p62 (left panel, green, middle left), Phalloidin (left panel, orange, middle right) and pseudocoloured CellProfiler overlay images (left panel, merged, right) and quantification of p62 puncta per cell (right panel) of MMTV-PyMT WT (black bars) and MMTV-PyMT IK KO (red bars). Bars represent average ±SEM, n = 4 with ***p ≤ 0.001, unpaired t-test. Scalebar = 10 µm. E Summarizing scheme of autophagic pathway and the consequence of IK channel deficiency (red arrows). AKT: Protein kinase B; mTORC1: Mammalian target of rapamycin complex 1; TSC1/2: Tuberous sclerosis proteins 1/2. F Representative FRET (left panel, yellow, left), CFP (left panel, cyan, middle) and pseudocoloured FRET-ratio images (left panel, 16-colors, right) and basal FRET-ratio signals (right panel) of MMTV-PyMT WT (black bar) and MMTV-PyMT IK KO cells (red bar) expressing AKTAR, a FRET-based AKT indicator (AKTact). Bars represent average ±SEM, n = 5, unpaired t-test. Scalebar = 20 µm. G Representative FRET (left panel, yellow, left), CFP (left panel, cyan, middle) and pseudocoloured FRET-ratio images (left panel, 16-colors, right) and basal FRET-ratio signals (mean of 5 min measurement, right panel) of MMTV-PyMT WT (black bar) and MMTV-PyMT IK KO cells (red bar) expressing TORCAR, a FRET-based mTORC1 indicator (mTORC1act). Bars represent average ±SEM, n = 6, unpaired t-test. Scalebar = 20 µm. H Summarizing scheme of metabolic pathway and the influence of IK channel deficiency (red arrows). Lack of IK results in a decreased Ca2+ load in multiple cellular compartments affecting mitochondrial ATP-production. Consistent with lower glycolytic activity in MMTV-PyMT IK KO cells, these events trigger AMPK activation, leading to compensatory induction of autophagy. Together with the postulated lower energy consumption, autophagy explains the enhanced cytosolic ATP level in IK-depleted cells. Figure created using BioRender.
