BI-1-deficient mice have reduced autophagy. (A) Protein levels of p62 were assessed by immunoblot analysis of hearts from wild-type (wt) and BI-1 knockout (ko) mice treated with rapamycin for various times as indicated (in hours). Lysates were normalized for total protein content and analyzed by SDS-PAGE/immunoblotting using anti-p62 or anti-tubulin antibodies. Wild-type (wt) and BI-1 knockout (ko) livers (B) and lungs (C) were collected after 24 h of injection with rapamycin. Tissues lysates were analyzed by immunoblotting as above using anti-LC3, anti-p62, and anti-tubulin antibodies. (D) Wild-type (wt) and BI-1 knockout (ko) mice were injected with rapamycin. After 24 h, hearts were dissected, processed, and examined by transmission electron microscopy. From 20 images each for wild-type (wt) and knockout specimens, the total number of autophagosomes was determined using ImageJ software. Mice were injected with 1 mg of rapamycin per kilogram of body weight. Horizontal bars indicate mean. Data are statistically significant by t-test (P = 0.009). (E) Female BALB/c nu/nu mice were injected subcutaneously with H322M cells (5 × 10⁶) containing scrambled control or BI-1 shRNA vectors. Tumor volumes were measured over time (mean ± SD; n = 10). (F) Seven weeks post-transplantation, animals were sacrificed, and tumors were excised and weighed (mean ± SD; n = 10 animals per group). (G) Tumor sections were analyzed for LC3 and p62 staining by quantitative immunohistochemistry. The overall low percentage of cells showing punctate LC3 immunostaining may reflect the antibody conditions employed, which were designed to avoid detection of diffuse cytosolic staining of nonautophagic cells.

BI-1 knockdown reduces autophagic flux. (A) Stably transduced H322M cells were cultured in standard rich medium or with serum- and glucose-deficient medium ([H] HBSS; [E] EBSS) alone or with various agents, including rapamycin (R; 25 μg/mL), thapsigargin (TG; 5 μM), and tamoxifen (TAM; 10 μM), for 16 h. Lysates were prepared, normalized for total protein content, and analyzed by SDS-PAGE/immunoblotting using anti-p62 and anti-tubulin antibodies. (B) H322M cells were stably transduced with recombinant shRNA lentiviruses targeting BI-1 or GFP. Relative levels of endogenous BI-1 mRNA were assessed by quantitative RT–PCR (expressing data as a percentage of control relative to cells transduced with GPF shRNA control vector). (C) H322M cells that were stably transduced with recombinant shRNA lentiviruses targeting BI-1 or GFP (control) were treated with LIs (20 mM NH₄Cl and 100 μM leupeptin) for either 2 h or 4 h, as indicated. Levels of LC3-I and LC3-II were analyzed by immunoblotting. Blots were reprobed with anti-β-actin antibody as a loading control. (D) Bands were quantified by densitometry, and measurements were used to calculate LC3 flux (mean ± SD; n = 3 experiments; P = 0.017).

BI-1 overexpression increases autophagic flux. (A) The relative levels of LC3-I and LC3-II were assessed by immunoblotting using lysates prepared from 293T cells transiently transfected with empty vector or BI-1-HA vector. Where indicated, cells were pretreated with bafilomycin (bafilo; 200 nM) or rapamycin (rapa; 25 μg/mL) for 16 h, or HBSS for 4 h. (B) The levels of p62 and LC3 were measured in HeLa cells in which BI-1 expression was conditionally driven using a doxycycline-inducible system. Cells were cultured with various concentrations (0, 50, 100, 250, 500, and 1000 ng/mL) for 12 h. (Left) Untreated cell. (Right) Rapamycin-treated cells (25 μg/mL for 12 h). Cell extracts were normalized for total protein content before analysis by SDS-PAGE/immunoblotting using anti-HA (to detect HA-BI-1), anti-p62, and anti-LC3 antibodies. Tubulin was used to verify equal protein loading. (C) HeLa cells in which BI-1 expression was conditionally driven using a doxycycline-inducible system (1 μg/mL) were treated with LIs (20 mM NH₄Cl and 100 μM leupeptin) for either 2 h or 4 h. Levels of LC3-I and LC3-II were analyzed by immunoblotting of cell lysates. β-Actin served as a loading control. (D) LC3-II bands were quantified by densitometry, and measurements were used to calculate LC3 flux. LC3 flux was quantified by dividing levels of LC3-II after 2 h of LI treatment per level of LC3-II without LI (mean ± SD; n = 3 independent experiments; P = 0.026).

BI-1 requires IRE1α but not autophagy for cytoprotective activity. (A) Wild-type (wt) and Atg7^−/− (knockout [ko]) MEFs were stably transduced with empty or BI-1-encoding lentiviruses. Cells were cultured under basal conditions (NT) or for 24 h with various cell stress agents ([TAM] 20 μM tamoxifen; [Rapa] 35 μg/mL rapamycin; [GSD] glucose and serum [FBS] deprivation; [TM] 10 μg/mL tunicamycin) in complete medium or nutrient-depleted medium (HBSS) for 8 h. Cells were then stained with annexin V–FITC and analyzed by FACS. Results are expressed as percentage of annexin V⁺ cells. (B) To verify defective autophagy in Atg7^−/− MEFs, LC3 levels were analyzed by immunoblotting. Tubulin was used as a loading control. (C) A clonogenic survival assay was performed using Atg7^−/− (ko) cells stably transduced with empty or BI-1-encoding lentivirus. Cells were cultured (200 cells per six-well plate) in nutrient-depleted medium (EBSS) for 4 h, then washed and cultured for an additional 10 d in complete medium. Cells were stained with crystal violet, and the number of colonies (>1 mm) per well was counted (mean ± SD; n = 3; P = 0.001 for EBSS-treated control vs. BI-1). (D) Saturated overnight cultures of BY4741 wild-type (wt) yeast or the indicated gene deletion strains containing a galactose-inducible Bax expression vector and either BI-1 or control plasmids were normalized to OD₆₀₀ = 1.0 and then serially diluted in increments of 1:10. Yeast cultures were spotted onto glucose (Bax-off) or galactose (Bax-on) plates and grown for 3 d at 30°C. (E) Ire1α^−/− (−) cells were stably transduced with either GFP or BI-1 lentiviruses. Cells were cultured for 24 h with various cell stress agents ([TG] 5 μM thapsigargin; [TM] 10 μM tunicamcyin; [Rapa] 35 μg/mL rapamycin; [GSD] glucose and FBS deprivation) or in nutrient-depleted medium (HBSS) for 8 h. Cell lysates were normalized for total protein content and subjected to immunoblot analysis to detect p62, Ire1, and tubulin (loading control). (F) Ire1α^−/− (knockout [ko]) or Ire1α^+/+ (wild-type [wt]) MEFs were stably transduced with either GFP or BI-1 lentiviruses and cultured with cell stress agents as in E. To assess cell death, cells were stained with annexin V–FITC, enumerating the percentage of annexin V⁺ (dead) cells by flow cytometry (mean ± SD; n = 3 independent experiments). (G) Clonogenic survival assays were performed using Ire1α^−/− MEFs stably transduced with GFP control or BI-1 lentiviruses. Cells were seeded (200 cells per six-well plate) in nutrient-depleted medium (EBSS) for 4 h and then cultured for an additional 10 d in nutrient-rich medium. Cells were stained with crystal violet, and the number of colonies (>1 mm) per well was counted (mean ± SD; n = 3 independent experiments). Differences between control and BI-1-overexpressing cells were not significantly different (P = 0.09 by t-test).

SERCA nullifies BI-1-mediated reduction of ER Ca²⁺. HeLa cells in which BI-1 expression was conditionally driven using a doxycycline-inducible system were cultured with (BI-1) or without (control [C]) 1 μg/mL doxycycline for 12 h. Cells were then loaded with the cytosolic Ca²⁺ probe Indo-1 AM (A) or the mitochondrial Ca²⁺ probe Rhod 2-AM (B) for 30 min at 37°C. After transfer to Ca²⁺-free PBS, cells were stimulated with thapsigargin (5 μM) and immediately analyzed using a microplate reader. Average fluorescent intensity was captured every 5 sec for a total time of 300 sec. We used a 405/485-nm emission ratio and 582 nm to measure Indo-1AM and Rhod-2 AM, respectively. (C,D) BI-1-inducible HeLa cells were either transiently transfected with control versus SERCA plasmids (C) or treated with DMSO versus SERCA agonist compound SB-6471 (10 μM) (D). Cells were then cultured with (to induce BI-1) or without doxycycline for 12 h prior to Indo1-AM loading. After transfer to Ca²⁺-free medium, thapsigargin (TG; 5 μM) was added (arrow), and the levels of cytosolic Ca²⁺ were measured every 5 sec for a total time of 300 sec. (E) Control and BI-1-overexpressing cells (BI-1) were transfected with the ER Ca²⁺ cameleon plasmid YC4.3ER for 48 h prior to analysis of ER Ca²⁺. (F) BI-1-inducible HeLa cells were either transiently transfected with control versus SERCA plasmids or treated with DMSO versus SERCA agonist compound SB-6471 (10 μM). Cells were then cultured with (to induce BI-1) or without doxycycline for 12 h prior to ER cameleon transfection and ER Ca²⁺ analysis. Results are expressed as emission ratio YFP/CFP.

BI-1 reduces mitochondria bioenergetics. (A) Activity of mitochondrial dehydrogenases was measured in control ([−] doxy) and BI-1 overexpressing ([+] doxy) HeLa cells. Values were normalized for the number of viable cells (trypan blue exclusion). Background absorbance was read at 690 nm and subtracted from readings at 440 nm (A440nm − A690nm). The difference between samples was significant (P < 0.05, by unpaired t-test). (B) Endogenous oxygen consumption rates in control (no doxy) and BI-1-overexpressing (doxy) cells were monitored by an oxymeter before (NT) and after exposure to oligomycin (2 μM) and FCCP (1 μM). Results are expressed as nanomoles of O₂ per minute per 1 × 10⁶ cells. Results for untreated control versus BI-1-expressing cells were statistically significant (P = 0.0015). (C) RCR in control ([−] doxy) and BI-1 overexpressing ([+] doxy) cells was calculated by dividing O₂ consumption before and after addition of oligomycin. The efficiency of oxidative phosphorylation coupling was comparable in control and BI-1-overexpressing cells. (D) ATP levels were measured in control versus BI-1-overexpressing cells using a bioluminescence method. Data represent relative luminescence units (RLUs) per 10⁶ viable cells (mean ± SD; n = 3; P = 0.002. (E) Levels of extracellular lactate were measured in control ([−] doxy) and BI-1-overexpressing ([+] doxy) cells. Results are represented as nanomoles of lactate per well (mean ± SD; n = 3; P = 0.021. (F) Lysates from control versus BI-1-overexpressing cells were normalized for total protein content and subjected to immunoblot analysis to detect AMPK, phospho-AMPK, acetylCoA carboxylase (ACC), and phospho-acetylCoA carboxylase (P-ACC). Anti-HA antibody was used to detect expression of BI-1-HA protein. Equivalent sample loading was shown by monitoring tubulin levels.

BI-1 requires IP₃R to modulate ATP levels and induce autophagy. (A) Wild-type (wt) and TKO IP₃R DT40 cells were stably infected with either control or BI-1 viruses. Where indicated, cells were treated with the LIs NH₄Cl (20 mM) and leupeptin (10 μM) for either 2 h or 4 h. (B) Levels of LC3-II were quantified by densitometry to measure LC3 flux. (C) ATP levels were measured in wild-type (wt) and TKO IP₃R DT40 cells stably transduced with either GFP or BI-1 lentiviruses. Data represent relative luminescence units (RLUs) per 10⁶ viable cells (trypan blue exclusion) of three independent measurements. (D) By increasing ER Ca²⁺ leakage via the IP₃R, BI-1 reduces the amount of Ca²⁺ at the microdomains where ER and mitochondria are in close proximity. Consequently, low mitochondrial Ca²⁺ levels reduce activity of the TCA cycle with reduction of O₂ consumption and ATP production. Low ATP levels trigger a cascade of signal transduction events that activates autophagy.