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

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Manufacturing of recombinant adeno-associated viral vectors for clinical trials

doi: 10.1038/mtm.2016.2

Figure Lengend Snippet: Institutions and rAAV GMP manufacturing technologies

Article Snippet: Targeted Genetics Corporation, Seattle, WA , wtAd5 Infection , HEK293 Producer HeLa Cell line WAVE and stir Tank bioreactors , Depth filtration, Benzonase, Ion exchange, UF/TFF, Chrom step, Heat inactivation, Chrom step, NanofiltrationPolishingFormulation , 1, 2 , , .

Techniques: Purification, Transfection, Flocculation, Lysis, Filtration, Plasmid Preparation, Concentration Assay, Affinity Chromatography, Sonication, Clarification Assay, Chromatography, Diafiltration Assay, Infection, Ion Exchange Chromatography

Journal: PLOS ONE

Article Title: Helicase-like transcription factor (HLTF) -deleted CDX/TME model of colorectal cancer increased transcription of oxidative phosphorylation genes and diverted glycolysis to boost S-glutathionylation in lymphatic intravascular metastatic niches

doi: 10.1371/journal.pone.0291023

Figure Lengend Snippet: Statistics for spatial transcriptomics outcome for Visium_FFPE_ HLTF -/- CDX in ID Hltf KO mice (A and B) and HLTF +/+ CDX in ID Hltf KO (C and D).

Article Snippet: Thus, System Biosciences (Palo Alto, CA) produced a custom stable cell line with shRNA sequence targeting of HLTF [ ].

Techniques: Sequencing

Journal: PLOS ONE

Article Title: Helicase-like transcription factor (HLTF) -deleted CDX/TME model of colorectal cancer increased transcription of oxidative phosphorylation genes and diverted glycolysis to boost S-glutathionylation in lymphatic intravascular metastatic niches

doi: 10.1371/journal.pone.0291023

Figure Lengend Snippet: A. The relative abundance of each microbial species was estimated based on the depth and coverage of sequencing across every available reference genome. The top 10 species are provided in bar graph format for individuals subdivided into two cohorts, i.e. ID male mice that were either Hltf +/+ (control) or Hltf KO. B. The Shannon Diversity Index was used to measure the diversity of taxa within the samples and between the cohorts. Box-and-whisker plots summarize numerical taxonomic (family) data based on quartiles. The inner quartile range < 2 indicates the values are not significantly different despite the differences in median values of 2.99 and 2.63, respectively, for Hltf +/+ and Hltf KO.

Article Snippet: Thus, System Biosciences (Palo Alto, CA) produced a custom stable cell line with shRNA sequence targeting of HLTF [ ].

Techniques: Sequencing, Control, Whisker Assay

Journal: PLoS ONE

Article Title: Loss of Mll3 Catalytic Function Promotes Aberrant Myelopoiesis

doi: 10.1371/journal.pone.0162515

Figure Lengend Snippet: Shown is A , the total number of BM cells, and the percentages of B , B cells and C , Gr1 + Mac1 + myeloid cells, where each point on the graph represents one mouse. Values in panels B and C are expressed as percentages of total cells. D , Representative H&E stains from one individual Mll3 +/+ and Mll3 Δ/Δ mouse of sternum sections taken at 400X magnification. Images depict the entire field of view (top panels) and a zoomed-in view (bottom panels). Scale bars are 50μm. E-I , Similar to panels B and C , except that the percentages of LSK cells, CLP, CMP, MEP, and GMP are shown, respectively. B, C, I , *p<0.05, **p<0.01, ***p<0.005 as determined by the Student’s t -test. G , *p<0.05 as determined by the Mann-Whitney test.

Article Snippet: Control (reference clone) and MLL3 -targeted clones of the human chronic myeloid leukemia cell line KBM7, engineered via a gene-trap system, were purchased from Haplogen (Vienna, Austria) [ ].

Techniques: MANN-WHITNEY

Journal: PLoS ONE

Article Title: Loss of Mll3 Catalytic Function Promotes Aberrant Myelopoiesis

doi: 10.1371/journal.pone.0162515

Figure Lengend Snippet: Absolute gene expression profiling adapted from Gene Expression Commons evaluating Mll3 expression across 39 different hematopoietic cell populations in the mouse BM, spleen, and thymus. Shown are the normalized values of Mll3 expression activity for several cell populations from 2–4 biological replicates. Positive values indicate high Mll3 expression compared to the common reference, and negative values denote relatively low Mll3 expression in comparison to the common reference. Error bars represent mean + SD. HSC, hematopoietic stem cell; Gra, granulocyte; Fr, fraction; Mz, marginal zone; Fo, follicular.

Article Snippet: Control (reference clone) and MLL3 -targeted clones of the human chronic myeloid leukemia cell line KBM7, engineered via a gene-trap system, were purchased from Haplogen (Vienna, Austria) [ ].

Techniques: Gene Expression, Expressing, Activity Assay, Comparison

Journal: PLoS ONE

Article Title: Loss of Mll3 Catalytic Function Promotes Aberrant Myelopoiesis

doi: 10.1371/journal.pone.0162515

Figure Lengend Snippet: The percentage of total cells (top) and absolute number (bottom) of A , B cells, B , T cells, and C , Gr1 + Mac1 + myeloid cells is shown, where each point represents one mouse. D , Representative H&E stains of spleen sections (50X magnification, top panels; 400X magnification, middle panels) from one individual Mll3 +/+ and Mll3 Δ/Δ mouse. Images in the bottom panels are zoomed-in views of the middle panels. Scale bars are 500μm (top panels) and 50μm (middle panels). E , Quantification of splenic follicle size. Each point on the graph represents the average follicle size (black, ≥10 follicles measured; red, <10 follicles measured) for one mouse. *p<0.05, **p<0.01 as determined by the Student’s t -test.

Article Snippet: Control (reference clone) and MLL3 -targeted clones of the human chronic myeloid leukemia cell line KBM7, engineered via a gene-trap system, were purchased from Haplogen (Vienna, Austria) [ ].

Techniques:

Journal: PLoS ONE

Article Title: Loss of Mll3 Catalytic Function Promotes Aberrant Myelopoiesis

doi: 10.1371/journal.pone.0162515

Figure Lengend Snippet: Shown is the percentage of total cells (top) and absolute number (bottom) of A , B cells, B , T cells, and C , Gr1 + Mac1 + myeloid cells. D , Similar to panels A-C , except the total numbers of CD4 + (left) and CD8 + (right) T cells are shown. Each point represents one mouse. B-C , *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 as determined by the Student’s t -test. D , *p<0.05, **p<0.005 as determined by the Mann-Whitney test. E , Representative H&E stains (top panels) and IHC for CD3 (bottom panels) from two individual Mll3 +/+ and Mll3 Δ/Δ mice of LN sections taken at 50X magnification. Scale bars are 500μm.

Article Snippet: Control (reference clone) and MLL3 -targeted clones of the human chronic myeloid leukemia cell line KBM7, engineered via a gene-trap system, were purchased from Haplogen (Vienna, Austria) [ ].

Techniques: MANN-WHITNEY

Journal: PLoS ONE

Article Title: Loss of Mll3 Catalytic Function Promotes Aberrant Myelopoiesis

doi: 10.1371/journal.pone.0162515

Figure Lengend Snippet: A , Relative MLL3 expression in human KBM7 cells as determined by qPCR. TaqMan probes (indicated in red) recognized exon junctions before (exons 18–19) and after (exons 39–40 and 55–56) the gene-trap (exon 38), indicated in green. B , Relative adhesion of KBM7 cells to fibronectin as measured in fluorescence units. Shown is the average of technical replicates from four individual experiments + SEM. C , The number of KBM7 cells that migrated across a trans-well membrane in response to FBS after 24h. Data are the combination of technical replicates from four individual experiments + SD. Ref, control gene-trap; MLL3, MLL3 -targeted gene-trap. *p<0.05, **p<0.01 as determined by the Mann-Whitney test.

Article Snippet: Control (reference clone) and MLL3 -targeted clones of the human chronic myeloid leukemia cell line KBM7, engineered via a gene-trap system, were purchased from Haplogen (Vienna, Austria) [ ].

Techniques: Expressing, Fluorescence, Membrane, Control, MANN-WHITNEY