PMC

(A–B) Bid-deficient hepatocytes were incubated with 10% serum alone (control) or with ionomycin (1 µM) or thapsigargin (TG, 0.1 µM) for the indicated time. Data (mean± SD) represented at least two independent experiments (A). p< 0.01 (ionomycin vs control and vs TG, 48 hr). Immunoblot assay was conducted with the indicated antibodies (B). (CA–D) Wild type hepatocytes were incubated with 10% serum alone (control) or with TG (0.1 µM) or EGTA (0.5 mM) for the indicated time. Data (mean± SD) represented at least two independent experiments (C). p<0.01 (control vs TG and vs EGTA, 48 hr). Immunoblot assay was conducted with the indicated antibodies (D). Asterisk indicates non-specific bands (B, D).

Wild type (Bid +/+) and Bid-deficient (Bid −/−) hepatocytes were transfected with the ER-targeted calcium sensor, D1ER (A–C) or the cytosol-targeted sensor, YC2.3 (D–E) for 48 hours. The ER expression pattern of D1ER (A) and cytosol expression pattern of YC2.3 (D) in wild type cells were shown. Scale bar: 10 µm. The FRET (YFP) and CFP signals were then recorded at the indicated time before and after TG (0.4 µM) stimulation. The ratio of FRET/CFP (mean± SD) was determined (B, E) and converted to the concentration of calcium in the case of D1ER (C).

(A) Wild type (WT), Bax-deficient (Bax −/−) and Bid/Bax-deficient (Bid/Bax −/−) hepatocytes were incubated with 10% serum for the indicated time. Data (mean± SD) represented at least three independent experiments. p<0.01 (WT vs Bax −/− and vs Bid/Bax −/−, 48 hr). (B) Hepatocytes of different genotypes were incubated with serum alone or with serum plus ionomycin (1 µM) for the indicated time. Immunoblot assays were conducted with the indicated antibodies. Asterisk indicates non-specific bands. (C–D) Bax-deficient (C) or Bid/Bax-deficient (D) hepatocytes were incubated with 10% serum alone or with ionomycin (1 µM, C–D) or thapsigargin (TG, 0.1 µM, C) for the indicated time. Data (mean± SD) represented at least three independent experiments. p<0.01 (panel C: ionomycin vs control and vs TG, 48hr); p<0.01 (panel D: ionomycin vs control, 48 hr).

(A) Primary hepatocytes from wild type (Bid +/+) and Bid-deficient (Bid −/−) mice were cultured on cover slides without serum for overnight and then continuously cultured without or with 10% serum as indicated for 48 hours and with BrdU in the last 24 hours. Hepatocytes were fixed and stained with anti-BrdU (red) and Hoeschst 33342 (blue). Scale bar: 25 µm. (B) The percentage of nuclei with BrdU incorporation was determined. At least three hundreds of nuclei were counted for each condition. Data (mean± SD) represented four independent experiments. p<0.01 at 48 hr (WT vs KO). (C) Hepatocytes were harvested at the indicated time of serum stimulation and the total cell lysates were subjected to immunoblot assay with the indicated antibodies. Asterisk indicates non-specific bands.

(A–B) Bid-deficient hepatocytes were infected with adenoviral GFP, GFP-Bid and ER-targeted GFP-Bid-b5 for 24 hours. Immunoblot assay was conducted with an anti-GFP and an anti-Bid antibody (A). Fluorescence microscopic examination demonstrated the cellular localization of these constructs (B). Note the ER location pattern of GFP-Bid-b5 in the enlarged panel. Scale bar: 10 µm. (C–D) Bid-deficient hepatocytes were infected with adenoviral GFP, GFP-Bid and GFP-Bid-b5 as indicated for overnight in serum-free medium. Cells were then cultured in medium containing 10% serum for the indicated time with BrdU included in the last 24 hours. The percentages of nuclei with BrdU incorporation in GFP, GFP-Bid or GFP-Bid-b5-positive hepatocytes were determined (C). Data (mean± SD) represented at least three independent experiments. p<0.01 (GFP-Bid-b5 vs GFP and vs GFP-Bid, 24 hr); p<0.05 (GFP-Bid vs GFP, 48 hr); p<0.01 (GFP vs GFP-Bid and vs GFP-Bid b5, 72 hr). Lysates were collected and immunoblot assay was conducted with indicated antibodies (D). (E). Subcellular fractions from wild type livers were subjected to immunoblot assay with the indicated antibodies. 14-3-3ε, VDAC and calnexin were used as controls for the cytosol (S10, S100), mitochondrial (P10) and ER (P100) fractions, respectively. Asterisks indicate non-specific bands. (F). The ER-enriched P100 fraction obtained from wild type mouse livers was either untreated (control, lane 1) or treated with 0.1M Na₂CO₃ (pH 11.5) on ice for 30 min, following by centrifugation (100,000 × g) for 30 min. Both the pellet (membrane fraction, lane 2) and the supernatant (lane 3) of alkali-treated sample were recovered for immunoblot assay. (G). Wild type (WT) hepatocytes were cultured in medium with 10% serum for 24 hours and then harvested. Subcellular fractions of S100 and P100 (Lane 1–2) were analyzed by immunoblot. The total lysate of bid−/− MEF (Lane 3) was used as a control for Bid signal.