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(A) Bcl-xL was cotransfected into HeLa cells with empty vector (IP negative control), Beclin 1-Flag, Beclin 1-Flag/HA-Bim(EL), Beclin 1-Flag/HA-Puma, Beclin 1-Flag/HA-Noxa, or Beclin 1-Flag/Bad. Anti-Flag (M2) was used for immunoprecipitation, and proteins were detected with anti-Bcl-xL, anti-HA, or anti-Bad, anti-Flag (Rabbit).(B) HA-Bim (wild-type) was cotransfected into Bax/Bak double-knockout (DKO) mouse embryonic fibroblasts (MEFs) with empty vector (IP negative control) or Beclin 1-Flag. Immunoprecipitations were performed as in (A).(C) Beclin 1-Flag was cotransfected into Bax/Bak DKO MEFs with empty vector (IP negative control) or HA-Bim (wild-type). Anti-HA was used for immunoprecipitation. ∗, antibody heavy chain.(D) HeLa cell lysates were subjected to GFP antibody (negative control) and anti-Bim antibody immunoprecipitation. Proteins were detected with anti-Beclin 1 and anti-Bim. ∗∗, antibody heavy chain; ∗, antibody light chain.(E) Bcl-xL was cotransfected into HeLa cells with empty vector (IP negative control), wild-type HA-Bim(EL), or HA-Bim L152E F159E [Bim(EL)EE]. Anti-Bim was used for immunoprecipitation. ∗, antibody light chain.(F) HeLa cells were transfected with vector, wild-type HA-Bim, or HA-BimEE. Lysates were probed with the indicated antibodies. HeLa cells treated with staurosporine (STS) for 5 hr were a positive control. Note that wild-type Bim causes apoptosis, which reduces its levels compared to BimEE.(G) Vector, wild-type Bim(EL), or Bim(EL) L152E F159E (BimEE) was transfected into HeLa cells. Transfection efficiency was >90%. After 20 hr, cell viability was determined by measurement pf ATP levels. Data are shown as mean ± SD. ∗∗∗p < 0.001.

(H) HeLa cells were transfected with vector/GFP (3:1), wild-type HA-Bim/GFP (3:1), or HA-BimEE/GFP (3:1) (to ensure that Bim-transfected cells also contain GFP). After 16 hr, cells were stained with Annexin V-APC and analyzed by FACS. The percentage of APC and GFP double-positive cells/GFP-positive cells are shown. Data are shown as mean ± SD. ∗∗∗p < 0.001.

(I) Bim(EL)/Bcl-xL were cotransfected into HeLa cells with empty vector (IP negative control) or Beclin 1-Flag; Bim(EL)-EE/Bcl-xL were also cotransfected into HeLa cells with Beclin 1-Flag. Anti-Flag (M2) was used for immunoprecipitation. Data are shown as mean ± SD. NS, not significant.

(J) BimEL-EE (EL)/vector (IP negative control), BimEL-EE (EL)/Beclin 1-Flag, BimL-EE (L)/Beclin 1-Flag, and BimS-EE (S)/Beclin 1-Flag were transfected into HeLa cells. Anti-Flag was used for immunoprecipitation. Data are shown as mean ± SD. ∗∗∗p < 0.001; NS, not significant.

(A) Bim−/− and Bim+/+ MEFs were cultured in 6-well dishes. Cells were treated with vehicle or Bafilomycin A1 (Baf) for 4 hr. Blots were probed with the indicated antibodies. LC3-II/tubulin in wild-type control MEFs is set as 1. The relative value of LC3-II/tubulin in Bim-knockout MEFs is shown (n = 5). Data are mean ± SD. ∗∗∗p < 0.001.

(B) HeLa cells were transfected with control siRNA and Bim siRNA. After 48 hr, cells were treated with vehicle or Baf for 4 hr. Blots were probed with the indicated antibodies and analyzed as described in (A). ∗∗∗p < 0.001.

(C) Bim−/− and Bim+/+ MEFs were transfected with GFP-LC3. The percentages of cells with GFP-LC3 vesicles were assessed. Data are shown as mean ± SD. ∗∗p < 0.01.

(D) HeLa cells were transfected with control plasmid (pMKO)/GFP-LC3 or pMKO-BimshRNA/GFP-LC3 (to ensure that GFP-LC3-positive cells also contain BimshRNA or control plasmid, the ratio of pMKO or pMKO-BimshRNA/GFP-LC3 is 3:1). The percentages of cells with GFP-LC3 vesicles were assessed. Data are shown as mean ± SD. ∗∗∗p < 0.001.

(E) HeLa cells were transfected with control plasmid (pMKO) or pMKO-BimshRNA. Blots were probed with the indicated antibodies. Data are shown as mean ± SD. ∗∗∗p < 0.001.

(F) HeLa cells were transfected with control plasmid (pMKO)/GFP-PX or pMKO-BimshRNA/GFP-PX (3:1). The percentages of cells with GFP-vesicles were assessed. Data are shown as mean ± SD. ∗∗p < 0.01.

(G) DFCP1-GFP cells were transfected with control siRNA or Bim siRNA. The numbers of GFP puncta were assessed. Data are shown as mean ± SD.∗p < 0.05.

Images represent DFCP1-GFP vesicles in cells transfected with control siRNA and Bim siRNA. Arrows mark DFCP1-GFP puncta.

(H) HeLa cells were treated with control siRNA or Bim siRNA. Blots were probed with the indicated antibodies. Data are shown as mean ± SD. ∗∗p < 0.01.

(I) Bim(EL)/control siRNA, Bim(EL)/Bim siRNA, Bim(EL)EE/control siRNA, Bim(EL)EE/Bim siRNA, vector/control siRNA, and vector/Bim siRNA were transfected into HeLa cells. Blots were probed with the indicated antibodies. Data are shown as mean ± SD. ∗∗p < 0.01.

(J) HeLa cells were transfected with control plasmid or Bim(EL)EE. Blots were probed with the indicated antibodies. Data are shown as mean ± SD. ∗∗p < 0.01.

(K) Bax/Bak DKO MEFs were transfected with control plasmid /GFP-LC3, wild-type Bim(EL)/GFP-LC3 (3:1), or Bim (EL) EE/GFP-LC3. The percentages of cells with GFP-LC3 vesicles were assessed. Data are shown as mean ± SD. ∗∗p < 0.01.

(L) Two independent control vector (pcDNA3) or Bim(EL)EE stably expressing HeLa cell clones were transfected with GFP-LC3 respectively. The percentages of cells with GFP-LC3 vesicles were assessed. Data are shown as mean ± SD. ∗∗∗p < 0.001.

(M) HeLa cells were transfected with control plasmid/GFP-PX or Bim(EL)EE/GFP-PX (3:1). The percentages of cells with GFP-vesicles were assessed. Data are shown as mean ± SD. ∗∗p < 0.01. Fluorescent microscope images show GFP-PX vesicles.

(A) Lysates from spleen-derived T cells of wild-type (Bim+/+) and Bim-knockout (Bim−/−) mice were immunoblotted and probed with indicated antibodies. Statistics were from three knockout and control mice. Data are shown as mean ± SD. ∗∗∗p < 0.001.

(B) T cells from Bim+/+ and Bim−/− mice were cultured with/without Baf for 4 hr preceding harvest, immunoblotted, and probed with the indicated antibodies.

(C) Spleen-derived T cells from Bim+/+ or Bim−/− mice were stained with rabbit anti-LC3 antibody. Representative confocal images and quantification from triplicates (n = 100-110) are shown. Data are shown as mean ± SD. ∗∗∗p < 0.0001.

(D) Bim+/+ and Bim−/− mouse liver lysates were immunoblotted and probed with the indicated antibodies (n = 3). Data are shown as mean ± SD. ∗∗p < 0.01.

(E) Bim+/+ and Bim−/− mouse liver lysates were immunoblotted and probed with the indicated antibodies (n = 3). Data are shown as mean ± SD. ∗∗∗p < 0.001.

(F) RNA from Bim wild-type or Bim-knockout mouse livers was analyzed by qRT-PCR for p62/beta-actin mRNA. The mean ± SD from three mice per group is shown. NS, not significant.

(A) HeLa cells were treated with control siRNA, Bim siRNA + control siRNA, control siRNA + Beclin 1 siRNA, or Bim siRNA + Beclin 1 siRNA. Blots were probed as indicated. Data are shown as mean ± SD. ∗∗p < 0.01.

(B) HeLa cells were treated with control siRNA, Bim siRNA + control siRNA, control siRNA + Beclin 1 siRNA, or Bim siRNA + Beclin 1 siRNA. After 24 hr, cells were transfected with GFP-PX for 24 hr. GFP-PX vesicles were assessed with a confocal microscope. Data are shown as mean ± SD. ∗∗p < 0.01. Arrows label cells with increased number/size of GFP-PX vesicles.

(C) GFP-mRFP-LC3 HeLa cells were treated with control, Bim siRNA, or Bcl-2 siRNA and then analyzed with Cellomics microscope. Data are shown as mean ± SD. ∗∗∗p < 0.001.

(D) GFP-mRFP-LC3 stable HeLa cells were treated with control siRNA, Bim siRNA + control siRNA, control siRNA + Beclin 1 siRNA, or Bim siRNA + Beclin 1 siRNA and then analyzed with Cellomics microscope and matching confocal images are shown. Data are shown as mean ± SD. ∗∗∗p < 0.001.

(A) Bim(EL)EE/empty vector (IP negative control) or Bim(EL)EE/Beclin 1-Flag (in duplicate) were transfected into HeLa cells. After 20 hr, one set of cells with Bim(EL)EE/Beclin 1-Flag were starved in HBSS for 2 hr. Anti-Flag antibody (M2) was used for immunoprecipitation, and blots were probed as indicated. Data are shown as mean ± SD. ∗∗∗p < 0.001.

(B) Panel i: HeLa cells were treated with control siRNA or Bim siRNA. After 48 hr, one set of transfections were starved in HBSS for 2 hr. Blots were probed as indicated. LC3-II/tubulin ratio of siRNA-transfected cells is set as 1 (n = 3). Data are shown as mean ± SD. ∗∗∗p < 0.001; NS, not significant. Panel ii: HeLa cells were treated with control siRNA or Bim siRNA. After 48 hr, one set of transfections were treated with Baf for 2 hr; one set of transfections were starved and treated with Baf for 2 hr. Blots were probed as indicated and analyzed as in (Bi). ∗∗p < 0.01; NS, not significant.

(C) HeLa cells were transfected with control plasmid /GFP-LC3 (3:1), or Bim(EL)EE/GFP-LC3 (3:1). After 20 hr, one set of transfected cells were starved in HBSS for 2 hr and then fixed. The percentages of cells with GFP-LC3 vesicles were assessed. Data are shown as mean ± SD. ∗∗p < 0.01. NS, not significant.

(D) BimS-Beclin 1 binding is not sensitive to HBSS starvation. BimEL-EE (EL)/vector (IP negative control), BimEL-EE (EL)/Beclin 1-Flag (two replicates), BimL-EE (L)/Beclin 1-Flag (two replicates) or BimS-EE (S)/Beclin 1-Flag (two replicates) were transfected into HeLa cells. After 20 hr, one of EL-Beclin 1-Flag transfections, one of L-Beclin-Flag transfections and one of S-Beclin 1-Flag transfections were subjected to 2 hr HBSS starvation. Anti-Flag (M2) was used for immunoprecipitation.

(E) HBSS starvation increases Bim T116 phosphorylation. Bim(EL)EE or BimEE(EL)-T116A were transfected into HeLa cells. After 20 hr, one set of Bim(EL)EE transfections and one of Bim(EL)EE-T116A were starved for 2 hr in HBSS. Blots were probed with the indicated antibodies.

(F) Bim T116 phosphorylation disables Bim-Beclin 1 interaction. Bim(EL)EE /vector (IP negative control), Bim(EL)EE/Beclin 1-Flag (in duplicate), or Bim(EL)EE-T116E (phospho-mimic, designated as T116E here)/Beclin-Flag (in duplicate) were transfected into HeLa cells. After 20 hr, one set of Bim(EL)EE/Beclin 1-Flag transfections and one of Bim(EL)EE-T116E/Beclin 1-Flag transfections were starved for 2 hr in HBSS (Starv). Anti-Flag (M2) was used for immunoprecipitation.

(G) HeLa cells were transfected with control plasmid /GFP-LC3, Bim(EL)-EE/GFP-LC3 (3:1), or Bim(EL)-EE T116E/GFP-LC3 (3:1). After 20 hr, one set of transfected cells were starved in HBSS for 2 hr and the cells were then fixed. The percentages of cells with GFP-LC3 vesicles were assessed. Data are shown as mean ± SD. ∗∗p < 0.01; NS, not significant.

(A) Bim-147 does not bind to Beclin 1. Beclin 1-Flag/vector (IP negative control), Beclin 1-Flag/Myc-BimEL-1-147aa (Myc-147), or Beclin 1-Flag/Myc-BimEL-ΔBH3 (Myc-ΔBH3) were transfected into HeLa cells. Anti-Myc (Rabbit) was used for immunoprecipitation.

(B) Bim-147 is competent for LC8 binding. LC8/vector (IP negative control), LC8/Myc-BimEL-1-147aa (Myc-147), or LC8/Myc-BimEL-ΔBH3 (Myc-ΔBH3) were transfected into HeLa cells. Anti-Myc (Rabbit) was used for immunoprecipitation.

(C) Mutating LC8 binding consensus sites within Bim disrupts the Bim-LC8 interaction. Bim(EL)EE/vector (IP negative control), Bim(EL)EE/Myc-LC8, Bim(EL)EE-S109A, S113A, T114A (AAA)/ Myc-LC8, or Bim(EL)EE-T116E/ Myc-LC8 were transfected into HeLa cells. Anti-Myc (Rabbit) was used for immunoprecipitation. Data are shown as mean ± SD. ∗∗∗p < 0.001.

(D) Mutating LC8 binding consensus sites within Bim disrupts the Bim-Beclin 1 interaction. Bim(EL)EE/vector (IP negative control), Bim(EL)EE/Beclin 1-Flag, Bim(EL)EE-S109A, S113A, T114A (AAA)/Beclin 1-Flag, or Bim(EL)EE-T116E/Beclin 1-Flag were transfected into HeLa cells. Anti-Flag (M2) was used for immunoprecipitation. Data are shown as mean ± SD. ∗∗∗p < 0.001.

(E) Bim-S does not bind to LC8. BimEL-EE (EL)/vector (IP negative control), BimEL-EE (EL)/Myc-LC8, BimL-EE (L)/Myc-LC8, or BimS-EE (S)/Myc-LC8 were transfected into HeLa cells. Anti-Myc (Rabbit) was used for immunoprecipitation.

(G) LC8 enhances Bim-Beclin 1 interaction in vitro. His-Bim, His-LC8 and GST-Beclin 1 were expressed in BL21(DE3) E. coli and purified. Two micrograms of His-Bim was combined with 2 μg GST without or with 1 μg His-LC8 (as controls); 2 μg His-Bim was combined with 2 μg GST-Beclin 1 without or with 1 μg His-LC8. The mixtures were incubated in buffer A for 3 hr. Glutathione beads were then used to pull down GST or GST-Beclin 1. The pulldown products were detected with anti-Bim and anti-Beclin 1.

(H) LC8 siRNA knockdown reduces the Bim-Beclin 1 interaction. Control siRNA or LC8 siRNA were transfected into HeLa cells. After 48 hr, HA-Bim(EL)EE/vector (IP negative control) or HA-Bim(EL)EE/Beclin 1-Flag was transfected into control siRNA transfected HeLa cells; HA-Bim(EL)EE/Beclin 1-Flag was transfected into LC8 siRNA-transfected HeLa cells. Anti-Flag (M2) was used for immunoprecipitation. Note that in the presence of BimEE, endogenous LC8 was pulled down by Beclin 1 when cells were treated with control siRNA. Data are shown as mean ± SD. ∗∗∗p < 0.001.

(A) HeLa cells were transfected with vector (vec) /GFP-LC3, BimEL-EE (EL)/GFP-LC3, BimL-EE (L)/GFP-LC3, BimS-EE (S)/GFP-LC3, or BimEL-EE-AAA (AAA)/GFP-LC3 (3:1). The percentages of cells with GFP-LC3 vesicles were assessed. Data are shown as mean ± SD. ∗∗∗p < 0.001; ∗∗p < 0.01; NS, not significant. Note that BimEL-EE-S109A, S113A, T114A is designated as BimEL-EE-AAA.

(B) Bim bridges the Beclin 1-LC8 interaction. Dynein light chain1 (Myc-LC8)/empty vectors (IP negative control), Myc-LC8/Beclin 1-Flag/empty vector, Myc-LC8/Beclin 1-Flag/BimEL-EE (EL), Myc-LC8/Beclin 1-Flag/BimL-EE (L), Myc-LC8/Beclin 1-Flag/BimS-EE (S), and Myc-LC8/Beclin 1-Flag/BimEL-EE-S109A, S113A, T114A (AAA) were transfected into HeLa cells. Anti-Flag (M2) was used for immunoprecipitation.

(C) Starvation reduces the ability of Bim to bridge the Beclin 1-LC8 interaction. Myc-LC8/empty vectors (IP negative control), Myc-LC8/Beclin 1-Myc/empty vector, or Myc-LC8/Beclin 1-Flag/HA-BimEL-EE (HA-Bim) (two replicates) were transfected into HeLa cells. After 20 hr, one set of cells with Myc-LC8/Beclin 1-Flag/HA-HA-Bim was starved in HBSS for 2 hr. Anti-Flag (M2) antibody was used for immunoprecipitation.

(D) HeLa cells were cultured with DMEM with 10% serum (Control) or starved (Starve) in HBSS for 2 hr. The fixed cells were stained with Beclin 1 and tubulin antibodies and analyzed by confocal microscopy. White boxes show the areas where Beclin 1 is enriched. Yellow boxes show enlarged areas.

(E) HeLa cells were treated with control siRNA or LC8 siRNA. After 24 hr, cells were split. Vector/GFP-LC3 or Bim(EL)EE/GFP-LC3 (3:1) were transfected into the control siRNA-transfected or LC8 siRNA-transfected cells. The percentages of cells with GFP-LC3 vesicles were assessed. Data are shown as mean ± SD. ∗∗p < 0.01; NS, not significant.

(F) HeLa cells were treated with control siRNA, Bim siRNA, or LC8 siRNA for 48 hr. Cells were then fixed in 37°C, 4% PFA for 10 min. Cells were stained with Beclin 1 and tubulin antibodies and analyzed by confocal microscopy. White boxes/triangle show areas where Beclin 1 is enriched. Yellow boxes show enlarged areas. Colocalizations were quantified from images in 12-15 cells with Volocity program (Colocalization coefficient Mx). Data are shown as mean ± SD. ∗∗∗p < 0.001.

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