Figure 5.

Mild and severe energy depletion affects the cytosolic and ER Ca²⁺ homeostasis. For measurement of cytosolic calcium dynamics HeLa cells were loaded with Fura-2 (+ Glu, n = 78 cells from four independent experiments; − Glu, n = 84 cells from three independent experiments; + 2-DG n = 73 cells from four independent experiments), for visualizing ER-calcium HeLa cells expressing D1ER were measured (+ Glu, n = 10 cells; − Glu, n = 14 cells; and + 2-DG n = 7 cells; each from three independent experiments). (a) Aligned dot plot and mean ± SD representing FRET ratio values of D1ER (i.e., [Ca²⁺]_ER, left) and Fura-2 ratio values (i.e., [Ca²⁺]_cyto, right) of unstimulated HeLa cells that were kept in the presence of 10 mM glucose (+ Glu, black), upon glucose starvation for 30 min (− Glu, blue), or cell treatment with 10 mM 2-DG for 30 min (+ 2-DG, red). *** significant versus + Glu p < 0.001, ♦♦♦ significant versus − Glu p < 0.001, Mann–Whitney U test; (b) Aligned dot plot and means ± SD showing the maximum change in ratio signals of D1ER (left) and Fura-2 (right) upon cell stimulation with ATP (100 µM) and 2,5-di-t-butyl- 1,4-benzohydroquinone (BHQ) (15 µM) in the absence of extracellular Ca²⁺ (i.e., EGTA), as also shown in panel c. * significant versus + Glu p < 0.05, *** significant versus + Glu p < 0.001, Mann–Whitney U test; (c) ER (upper panel) and cytosolic (lower panel) Ca²⁺ signals over time in the presence of 10 mM glucose (+ Glu control, black, left panels, n_ER = 10 cells, n_cyto = 78 cells), the absence of glucose (− Glu, blue, middle panels, n_ER = 14 cells, n_cyto = 84 cells), and upon cell treatment with 10 mM 2-DG (30 min prior to imaging experiments, + 2-DG red, right panels, n_ER = 7 cells, n_cyto = 73 cells). As indicated, cells were stimulated with a mixture of 100 µM ATP and 15 µM BHQ in the absence of extracellular Ca²⁺, i.e., in the presence of 0.1 mM EGTA. Subsequently, ATP and BHQ were removed and 2 mM Ca²⁺ was re-added via a perfusion system.