Journal: PLoS ONE

Article Title: Phosphorylation of the BNIP3 C-Terminus Inhibits Mitochondrial Damage and Cell Death without Blocking Autophagy

doi: 10.1371/journal.pone.0129667

Figure Lengend Snippet: (A) Western blot detection of immunoprecipitated BNIP3 using an α-PKA substrate antibody specific to the phosphorylated RRXS/T sequence, located at the BNIP3 C-terminus and adjacent to the transmembrane (TM) domain. Results shown for 4 cell types: (left to right) HEK 293 cells expressing exogenous BNIP3 (dimer, 60 kD), and A549, MDA-MD-231, and AU565 cells expressing endogenous BNIP3 (monomer, 30kD). Lane 1 of each Western blot contains the whole cell lysate (WCL). (B) LC-MS/MS analysis of BNIP3 phosphorylation in HEK 293 cells with normal or elevated cAMP (8-Bromo-cAMP), showing peptide coverage (gray) and phosphorylation sites (red). The TM domain is underlined. (C) Table of BNIP3 phosphopeptides identified by LC-MS/MS, showing the percent probability, ion charge, actual and observed masses, and mass error (Da and ppm) for each peptide. Peptides shown are from analysis of BNIP3 purified from HEK 293 cells with elevated cAMP levels. (D) Schematic of the BNIP3 protein sequence, showing each C-terminal mutation representing phosphomimetic or nonphosphorylated BNIP3. (E) Expression of BNIP3 phosphomutants from stable doxycycline-inducible HEK 293 Tet On cells, treated with doxycycline (Dox) for 48 hr. (F) Subcellular localization of BNIP3 phosphomutants, showing Western blot of cytosolic and mitochondrial fractions. (G) Alkaline extraction of mitochondria-associated proteins, showing alkaline extract of the mitochondrial pellet and the alkaline-resistant mitochondrial pellet from cells expressing each BNIP3 phosphomutant. All blots are representative of at least 3 independent experiments.

Article Snippet: HEK 293 Tet On cells (purchased from Clontech Laboratories, Inc., Cat # 631182) were maintained in α-MEM with 10% Tet system approved FBS (Clontech Laboratories, Inc.) and 100 μg/mL G418.

Techniques: Western Blot, Immunoprecipitation, Sequencing, Expressing, Liquid Chromatography with Mass Spectroscopy, Purification, Mutagenesis

Journal: PLoS ONE

Article Title: Phosphorylation of the BNIP3 C-Terminus Inhibits Mitochondrial Damage and Cell Death without Blocking Autophagy

doi: 10.1371/journal.pone.0129667

Figure Lengend Snippet: (A) Representative examples of mitochondrial morphology, examined via transmission electron microscopy. Black arrows denote healthy, elongated mitochondria and white arrows denote rounded, swollen mitochondria. Scale bar represents 2 μm. (B) Quantification of electron microscopy, showing the average mitochondrial area per field. At least 15 fields were examined per cell type. (C) Percent elongated mitochondria per field, quantified from at least 15 microscope fields per cell type. (D) Mitochondrial mass of HEK 293 cells expressing each BNIP3 mutant, measured by flow cytometric analysis of the mean fluorescence intensity (MFI) of Mitotracker Green FM. (E) Mitochondrial protein levels in HEK 293 cells expressing each BNIP3 mutant, monitored by detection of mitochondrially-encoded cytochrome c oxidase subunit II (MT-CO2). For bar graphs, significant differences between control cells (without BNIP3) and cells expressing each BNIP3 mutant are denoted by * p<0.05, ** p<0.01, and *** p<0.001; significant differences between cells expressing WT BNIP3 and either control cells or cells expressing each BNIP3 mutant are denoted by # p<0.05, ## p<0.01, and ### p<0.001; significant differences between complementary pairs of BNIP3 mutants are shown in brackets.

Article Snippet: HEK 293 Tet On cells (purchased from Clontech Laboratories, Inc., Cat # 631182) were maintained in α-MEM with 10% Tet system approved FBS (Clontech Laboratories, Inc.) and 100 μg/mL G418.

Techniques: Transmission Assay, Electron Microscopy, Microscopy, Expressing, Mutagenesis, Fluorescence

Journal: PLoS ONE

Article Title: Phosphorylation of the BNIP3 C-Terminus Inhibits Mitochondrial Damage and Cell Death without Blocking Autophagy

doi: 10.1371/journal.pone.0129667

Figure Lengend Snippet: (A) Representative examples of JC1 dual color fluorescence, examined by confocal microscopy. Red JC1 fluorescence is localized to polarized mitochondria whereas green fluorescence is independent of membrane polarization. Scale bar represents 10 μm. Red JC1 insets, denoted by white outlines, provide examples of the mitochondrial network in cells expressing each BNIP3 phosphomutant. Scale bar for inset is 5μm. Experimental controls included treatment of HEK 293 cells with Oligomycin A (Oligo A) or FCCP to hyperpolarize and depolarize mitochondria, respectively. (B) Mitochondrial membrane potential (ΔΨm), quantified as a ratio of red:green JC1 fluorescence by flow cytometry, where the same positive and negative controls were used to confirm efficacy of the assay (C) Levels of reactive oxygen species (ROS), measured by flow cytometric analysis of DHE fluorescence. (D) ROS levels, quantified by DCF fluorescence. (E) Mitochondrial ROS, measured as a ratio of MitoSox mean fluorescence intensity/Mitotracker mean fluorescence intensity for cells expressing each BNIP3 phosphomutant. All bar graphs represent the results observed in at least 3 independent experiments. Significant differences between control cells (without BNIP3) and cells expressing each BNIP3 mutant are denoted by * p<0.05, ** p<0.01, and *** p<0.001; significant differences between cells expressing WT BNIP3 and cells either lacking BNIP3 (None) or expressing each BNIP3 mutant are denoted by # p<0.05, ## p<0.01, and ### p<0.001; significant differences between pairs of complementary BNIP3 mutants are shown in brackets.

Article Snippet: HEK 293 Tet On cells (purchased from Clontech Laboratories, Inc., Cat # 631182) were maintained in α-MEM with 10% Tet system approved FBS (Clontech Laboratories, Inc.) and 100 μg/mL G418.

Techniques: Fluorescence, Confocal Microscopy, Expressing, Flow Cytometry, Mutagenesis

Journal: PLoS ONE

Article Title: Phosphorylation of the BNIP3 C-Terminus Inhibits Mitochondrial Damage and Cell Death without Blocking Autophagy

doi: 10.1371/journal.pone.0129667

Figure Lengend Snippet: (A) Representative images of GFP-LC3 puncta in HEK 293 cells expressing each BNIP3 mutant, examined via confocal microscopy. At least 50 cells were examined per cell type, in 3 independent experiments. Rapamycin (Rap) was used as a positive control. Scale bar represents 10 μm. (B) Quantification of the number of GFP-LC3 puncta per cell. (C) Western blot analysis of autophagic flux, where cells expressing each BNIP3 phosphomutant were treated without or with 50 nM Bafilomycin A1 (BAF). Blots are representative of 4 independent experiments. Two exposures of the LC3 Western blot are provided to show levels of LC3-II (short exposure) and LC3-I (long exposure). (D) Representative images of HEK 293 cells probed with Lysotracker Red, examined by confocal microscopy. (E) Quantification of the number of lysotracker puncta per cell, where a minimum of 30 cells were examined in 3 independent experiments. Scale bar represents 10 μm. (F) Quantification of mean lysotracker fluorescence intensity, measured by flow cytometry in 3 independent experiments. For bar graphs, significant differences between control cells (without BNIP3) and cells expressing each BNIP3 mutant are denoted by * p<0.05, ** p<0.01, and *** p<0.001; significant differences between cells expressing WT BNIP3 and either control cells or cells expressing each BNIP3 mutant are denoted by # p<0.05, ## p<0.01, and ### p<0.001; significant differences between complementary pairs of BNIP3 mutants are shown in brackets.

Article Snippet: HEK 293 Tet On cells (purchased from Clontech Laboratories, Inc., Cat # 631182) were maintained in α-MEM with 10% Tet system approved FBS (Clontech Laboratories, Inc.) and 100 μg/mL G418.

Techniques: Expressing, Mutagenesis, Confocal Microscopy, Positive Control, Western Blot, Fluorescence, Flow Cytometry

Journal: PLoS ONE

Article Title: Phosphorylation of the BNIP3 C-Terminus Inhibits Mitochondrial Damage and Cell Death without Blocking Autophagy

doi: 10.1371/journal.pone.0129667

Figure Lengend Snippet: (A) Levels of ROS, measured by the mean fluorescence intensity of DHE. HEK 293 control cells and cells expressing WT BNIP3 for 48 hr were treated with 8-Br-cAMP for 0, 2, or 4hr immediately prior to analyzing DHE fluorescence by flow cytometry. (B) Percent Annexin V positive cells undergoing the same 8-Br-cAMP treatment as described in (A). (C) Mitochondrial mass of HEK 293 cells cultured in normoxia or hypoxia for 48 hr and with or without 8-Br-cAMP treatment for the last 6 hr of normoxia/hypoxia. (D) Mitochondrial membrane potential of cells treated as described in (C). For each assay, a minimum of 30,000 events were collected per sample by flow cytometry. Data represents results from at least 3 separate experiments. Significant differences between untreated control cells (without BNIP3) and cells expressing WT BNIP3 are denoted by * p<0.05, ** p<0.01, and *** p<0.001; significant differences between treatment conditions are shown in brackets. (E) Phosphorylation of endogenous BNIP3 at T188 following nutrient deprivation for 0, 30, or 120 min in three cell types: A549, MDA-MB-231, and AU565 cells, detected by probing immunoprecipitated BNIP3 with an α-PKA substrate antibody. (F) Phosphorylation level of endogenous BNIP3 at T188 following hypoxia for 0, 6, 24, or 48 hr in three cell types: A549, MDA-MB-231, and AU565 carcinoma cells, detected by probing immunoprecipitated BNIP3 with an α-PKA substrate antibody. To account for the increased expression of BNIP3 during extended hypoxia, the relative level of T188 BNIP3 phosphorylation is provided below each lane. The phosphorylation level, which represents the ratio of BNIP3 detected by α-PKA substrate antibody/total BNIP3, is expressed relative to the 0 hr time point of each cell type.

Article Snippet: HEK 293 Tet On cells (purchased from Clontech Laboratories, Inc., Cat # 631182) were maintained in α-MEM with 10% Tet system approved FBS (Clontech Laboratories, Inc.) and 100 μg/mL G418.

Techniques: Fluorescence, Expressing, Flow Cytometry, Cell Culture, Immunoprecipitation