To test this hypothesis, we introduced into hepatocytes a specific fluorescent calcium probe that targets ER: D1ER (Fig. 3A). This new-generation calcium probe contains a calcium-binding domain and uses the FRET principle to quantify the level of calcium at the specific subcellular site where it is located.17 The

FRET signal is in proportion to the amount of calcium binding to this probe. Using this probe, we found that WT hepatocytes had a higher level of calcium in the ER than Bid-deficient hepatocytes (Fig. 3B,C). TG is an inhibitor of sarco/endoplasmic reticulum Ca2+ adenosine triphosphatase (SERCA), which is required for the reuptake of calcium leaking from the ER. Thus, TG causes a continuous decrease in the ER calcium level. We found no significant differences between WT and Bid-deficient hepatocytes buy Smoothened Agonist in terms of the kinetics of the TG-induced reduction, but WT cells consistently possessed a higher level of ER calcium

than Bid-deficient hepatocytes until virtually all ER calcium was depleted (Fig. 3B,C). These findings suggested KU-57788 that Bid was important for the maintenance of the ER calcium storage level. In the absence of Bid, this lower level of ER calcium storage would lead to a lower level of cytoplasmic calcium after release from the ER and subsequently a lower level of store-operated calcium entry (SOCE). This notion was supported by the measurement of the cytosol calcium level with another calcium probe, YC2.3, which is located in the cytoplasm17 (Fig. 3D). After TG treatment, the accumulation of calcium in the cytoplasm of Bid-deficient hepatocytes was much less than that in the WT hepatocytes (Fig. 3E). We also found that the ability of Bid to regulate ER calcium homeostasis was not limited to hepatocytes. Bid-deficient T lymphocytes12 and murine embryonic fibroblast cells (MEFs; Supporting Information Fig. 1A) also displayed a reduced proliferative response to mitogen stimulation. The introduction of YC2.3

into the WT MEFs indicated a calcium increase in the cytosol followed by TG treatment, which was significantly blunted in Bid-deficient MEFs (Supporting C-X-C chemokine receptor type 7 (CXCR-7) Information Fig. 1B). Using a different approach to calcium measurement with the fura-2-acetoxymethyl ester dye, we found that the basal level of cytosol calcium, the inositol-1,4,5-trisphosphate (InsP3)–sensitive calcium store, and the total intracellular calcium store were all reduced in Bid-deficient MEFs (Supporting Information Fig. 1C-F). Consistently, SOCE, as measured by the Mn2+ quenching method, was also reduced in Bid-deficient MEFs (Supporting Information Fig. 1G,H). Together, these findings from different approaches indicated that Bid could regulate ER calcium homeostasis. To determine that the calcium level was indeed responsible for the effects of Bid on hepatocyte proliferation, we included a calcium ionophore, ionomycin, in the culture medium together with the serum.