Figure 3
mTORC1 inhibitor rapamycin and ER stressor tunicamycin induce different patterns of ER-mitochondria communication.
(A) Example of the radial analysis in HeLa cells co-stained with ER-RFP (red) and MitoTracker Green (upper left). After image acquisition, cells were delineated and their area was determined (upper right). From this area, a hypothetical cell radius was calculated (lower left). According to this radius, 4 concentric circles were defined, using the middle of the nucleus as their centre (lower right). Analysis of fluorescence was then performed in the 5 regions defined by the circles. (B) Mitochondria-to-ER colocalization of control HeLa cells (con) or cells treated with tunicamycin (tun) or rapamycin (rap), calculated locally as the Manders’ coefficient within regions of the radial analysis (n = 3). (C) Upper panels: immunofluorescence of ER (anti-KDEL antibody, green) and mitochondria (mtHsp70, red) of control MDA-MB-231 cells (con) or cells treated with tunicamycin (tun) or rapamycin (rap), measured by confocal fluorescence microscopy. Lower panels: pseudocolor images indicating colocalization of both organelles. (D) Mitochondria-to-ER colocalization of images obtained as in (C) calculated locally as radial Manders’ coefficients (n = 3). Scale bars = 10 μm. Data are shown as the mean ± SEM. *P ≤ 0.05 compared with controls (con).
