Calcium release from the ER triggered by ionomycin and histamine. (A) Time-lapse montages of U2OS cells transfected with the ER calcium probe (ER-GCaMP6-150) and treated with DMSO (top), 4 µM ionomycin (middle), or 100 µM histamine (bottom). Time in seconds. Bar, 10 µm. Corresponds to Video 5. (B) Graph quantifying changes in ER calcium upon the treatments described in A; n = 12, 15, and 15 cells for DMSO, ionomycin, and histamine, respectively. Error bars represent SEM. (C) Effect of thapsigargin on ER calcium (black curves), actin burst (green curves), and mitochondrial calcium (red curves) before and after ionomycin stimulation. U2OS cells transfected with ER calcium probe and mApple-Ftractin. DMSO (top graph) or 1 µM thapsigargin (bottom graph) was applied at 0 s (bold line) and 4 µM ionomycin was applied at the time indicated by the dashed line; n = 10 cells (DMSO) or 13 cells (thapsigargin). Error bars represent SEM. (D) Effect of thapsigargin on histamine-induced cytoplasmic calcium and ER calcium changes. U2OS cells transfected with ER calcium probe and Cyto-R-GECO; n = 15 cells (DMSO) or 15 cells (thapsigargin). (E) Effect of thapsigargin on histamine-induced actin burst and ER calcium. U2OS cells were transfected with ER calcium probe and mApple-Ftractin; n = 10 cells (DMSO) or 11 cells (thapsigargin). Error bars represent SEM. (F) Effect of thapsigargin on histamine-induced mitochondrial calcium and ER calcium. U2OS cells transfected with ER calcium probe and mito-R-GECO; n = 12 cells (DMSO) or 14 cells (Thapsigargin). Error bars represent SEM. (G) Effect of thapsigargin on ionomycin-induced cytoplasmic calcium and ER calcium. U2OS cells transfected with both the Cyto-R-Geco and ER-GCaMP6-150; n = 17 cells (DMSO) or 16 cells (thapsigargin). Error bars represent SEM.