Figure 2
Low [Ca2+]ER leads to low [Ca2+]mito in HT1080/BI-1 cells.
(A) Mitochondrial and ER Ca2+ in parental HT1080, HT1080/Neo and HT1080/BI-1 cells after treatment with 5 μM thapsigargin (arrow) was analyzed with Rhod II AM fluorescence (upper) and erAEQ luminescence (lower), respectively. (B) HT1080/Neo and HT1080/BI-1 were loaded with Rhod II AM and treated with either 5 μg/ml tunicamycin or 1 μg/ml brefeldin A (arrow). Rhod II fluorescence was monitored over time. (C) Immunoblotting of HT1080/Neo and HT1080 cells expressing different amounts of BI-1 with antibodies against BI-1 and β-actin (upper). Mitochondrial Ca2+ in HT1080/Neo and BI-1–expressing HT1080 cells upon treatment with 5 μM thapsigargin was analyzed by Rhod II AM fluorescence. Specific colors indicate cells expressing different amounts of BI-1. (D) Immunoblotting of BI-1+/+ and BI-1-/- MEF cells with antibodies against BI-1 and β-actin (upper). BI-1+/+ and BI-1-/- MEF cells were incubated with Rhod II AM to analyze mitochondrial accumulation of Ca2+ after treatment with 5 μM thapsigargin (arrow) (left). BI-1+/+ and BI-1-/- MEF cells were transfected with erAEQ. The intensity of erAEQ luminescence was monitored after treatment with 5 μM thapsigargin (arrow) (right). Results represent the mean ± SEM of five independent experiments. Tg, thapsigargin; Tu, tunicamycin; Bre, Brefeldin A; erAEQ, ER-localized aequorin; HT, parental HT1080 cells; Neo, neomycin-resistant pcDNA3-transfected HT1080 cells (HT1080/Neo); BI-1, HA-BI-1-pcDNA3-transfected HT1080 cells (HT1080/BI-1).
