Figure 5. Effect of proteasome inhibitors on endoplasmic reticulum (ER) Ca²⁺ ([Ca²⁺]_ER).

A. Measurement of [Ca²⁺]_ER with mag-fura-2. Cultures were treated with MG-132 (2 μM) and then loaded with mag-fura-2 (see Methods) and [Ca²⁺]_ER was analyzed at the indicated time points after the start of the treatment period; images and data analysis were similar to that for fura-2 in Figures 1 and 3. n= 87-143 cells per time point. * indicates significant difference (P<0.05 by ANOVA) from sham-washed (time = 0) condition. B. Indirect measurement of [Ca²⁺]_ER with fura-2 and thapsigargin. Cultures were treated with MG-132 (2 μM) for 4 hr or 16 hr (darker and lighter gray lines respectively) or sham-washed for 4 hr (black line). Cultures were loaded with fura-2; before imaging, the media was changed to calcium free buffer. Baseline [Ca²⁺]_i was measured in Ca²⁺-free media and the cells were treated with thapsigargin (5 μM). After 5 min, thapsigargin was washed out, 1.8 mM calcium was added to the bathing media and [Ca²⁺]_i was measured for another 5 minutes. A representative trace is shown for each condition. Similar results were obtained for n= 8-10 cells each condition. MG-132 was present in treated cultures throughout the experiment.