Fig. 3.

Permeabilization of the plasma membrane prevents thapsigargin-induced ER lumen reduction. a Sequential images of digitonin (25 μg/ml)-treated HeLa cells loaded with BCECF-AM fluorescent dye. b, c Fluorescence ratio changes of HyPer-ER sensor 24 h after transfection in digitonin-permeabilized (red line) or intact (blue line) HeLa cells. Cells were pretreated with digitonin for 3 min and washed with intracellular medium as described in the “Materials and methods” section prior to the experiment. TG (200 nM, b) or puromycin (100 μM, c) were applied at 3 min of imaging as indicated by the arrow. Experiments were terminated by addition of 0.5 mM DTT. Traces represent average intensity ratios acquired from 14 to 34 cells of 4 independent experiments