Journal: The FASEB Journal

Article Title: Iron deficiency‐induced loss of skeletal muscle mitochondrial proteins and respiratory capacity; the role of mitophagy and secretion of mitochondria‐containing vesicles

doi: 10.1096/fj.201901815r

Figure Lengend Snippet: FIGURE 3 Iron chelation in myotubes results in indications toward increased mitophagy initiation. Presence of mitophagy events, ie, autophagosomal- enclosed mitochondria or lysosomal- enclosed mitochondria (black arrows) in myotubes exposed for 24 hours to control or 300 μM DFP (A). mRNA expression of mitophagy receptors Bnip3, Bnip3l, and Fundc1, and ubiquitin-mediated mitophagy effectors Pink1, and Park2 is depicted of myotubes exposed for 24 hours to control (white bars) or 300 μM DFP (black bars) (B). Protein expression of mitophagy receptors BNIP3, BNIP3L, and FUNDC1 is depicted of myotubes exposed to control (white bars) or 24 hours of 300 μM DFP (black bars) (C). Data are depicted as mean + SEM (n = 3), and significant differences are depicted *P < .05, **P < .01

Article Snippet: Subsequently, membranes were washed, blocked with 3% nonfat, dried milk (Campina, Amersfoort, UT, the Netherlands) in TBS-Tween-20 (0.05%) for 1 hour, washed, and incubated overnight at 4°C with different protein-specific primary antibodies against: BNIP3 (#3769S, RRID:AB_2259284, Cell Signaling Technology), BNIP3L (#12396, RRID:AB_2688036, Cell Signaling Technology), DNM1L (#8570, RRID:AB_10950498, Cell Signaling Technology), FUNDC1 (#sc-133597, RRID:AB_10609242, Santa Cruz Biotechnology), microtubule-associated protein light chain 3 (LC3)B (#2775, RRID:AB_915950, Cell Signaling Technology), Heatshock protein (HSP)70 (#NB11096425, RRID:AB_1262360, Novus), Flotillin-1 (FLOT1) (#610821, RRID:AB_398140, BD Biosciences), and oxidative phosphorylation (OXPHOS) complex subunits (#MS604, RRID:AB_2629281, MitoScience LLC) all diluted in 3% nonfat, dried milk or 5% bovine serum albumin in TBS-Tween-20.

Techniques: Control, Expressing, Ubiquitin Proteomics

Journal: The FASEB Journal

Article Title: Iron deficiency‐induced loss of skeletal muscle mitochondrial proteins and respiratory capacity; the role of mitophagy and secretion of mitochondria‐containing vesicles

doi: 10.1096/fj.201901815r

Figure Lengend Snippet: FIGURE 4 Inhibition of general autophagy initiation or lysosomal breakdown during iron chelation does not rescue loss of mitochondrial constituents. mtDNA/gDNA ratio (A), SDHB protein levels (B), BNIP3, BNIP3L, LC3BI, and LC3BII protein levels (C), are depicted of myotubes exposed for 24 hours to control (white bars), 300 μM DFP (black bars), control + 3-MA (light gray bars), or 300 μM DFP + 3-MA (dark gray bars). mtDNA/gDNA ratio (D), SDHB protein levels (E), BNIP3, BNIP3L, LC3BI, and LC3BII protein levels (F) are depicted of myotubes exposed for 24 hours to control (white bars), 300 μM DFP (black bars), control + BAFA1 (light gray bars), or 300 μM DFP + BAFA1 (dark gray bars). Autophagic flux (group-average fold change value without BAFA1 subtracted from the individual fold change values with BAFA1) is depicted for control (white bars) and 300 μM DFP (black bars) conditions (G). Data are depicted as mean + SEM (n = 3), and significant differences are depicted compared with control or indicated bars *P < .05, **P < .01, ***P < .001

Article Snippet: Subsequently, membranes were washed, blocked with 3% nonfat, dried milk (Campina, Amersfoort, UT, the Netherlands) in TBS-Tween-20 (0.05%) for 1 hour, washed, and incubated overnight at 4°C with different protein-specific primary antibodies against: BNIP3 (#3769S, RRID:AB_2259284, Cell Signaling Technology), BNIP3L (#12396, RRID:AB_2688036, Cell Signaling Technology), DNM1L (#8570, RRID:AB_10950498, Cell Signaling Technology), FUNDC1 (#sc-133597, RRID:AB_10609242, Santa Cruz Biotechnology), microtubule-associated protein light chain 3 (LC3)B (#2775, RRID:AB_915950, Cell Signaling Technology), Heatshock protein (HSP)70 (#NB11096425, RRID:AB_1262360, Novus), Flotillin-1 (FLOT1) (#610821, RRID:AB_398140, BD Biosciences), and oxidative phosphorylation (OXPHOS) complex subunits (#MS604, RRID:AB_2629281, MitoScience LLC) all diluted in 3% nonfat, dried milk or 5% bovine serum albumin in TBS-Tween-20.

Techniques: Inhibition, Control

Journal: The FASEB Journal

Article Title: Iron deficiency‐induced loss of skeletal muscle mitochondrial proteins and respiratory capacity; the role of mitophagy and secretion of mitochondria‐containing vesicles

doi: 10.1096/fj.201901815r

Figure Lengend Snippet: FIGURE 6 Iron chelation results in secretion of mitochondria-containing vesicles. Presence of mitochondrial- containing vesicles (black arrows) near the plasma membrane in iron chelation- exposed myotubes (A). Quantification of vesicles in myotubes subjected to iron chelation in presence of scrambled, BNIP3/ BNIP3L, or GABARAPL1 siRNA, three grids with 10 cells each were quantified (unless < 10 cells were available on the grid) per condition (B, C). Data are depicted as mean + SEM, and significant differences are depicted ***P < .001. siScr = Scramble siRNA, siB/B = BNIP3/BNIP3L siRNA, siGAB = GABARAPL1 siRNA

Article Snippet: Subsequently, membranes were washed, blocked with 3% nonfat, dried milk (Campina, Amersfoort, UT, the Netherlands) in TBS-Tween-20 (0.05%) for 1 hour, washed, and incubated overnight at 4°C with different protein-specific primary antibodies against: BNIP3 (#3769S, RRID:AB_2259284, Cell Signaling Technology), BNIP3L (#12396, RRID:AB_2688036, Cell Signaling Technology), DNM1L (#8570, RRID:AB_10950498, Cell Signaling Technology), FUNDC1 (#sc-133597, RRID:AB_10609242, Santa Cruz Biotechnology), microtubule-associated protein light chain 3 (LC3)B (#2775, RRID:AB_915950, Cell Signaling Technology), Heatshock protein (HSP)70 (#NB11096425, RRID:AB_1262360, Novus), Flotillin-1 (FLOT1) (#610821, RRID:AB_398140, BD Biosciences), and oxidative phosphorylation (OXPHOS) complex subunits (#MS604, RRID:AB_2629281, MitoScience LLC) all diluted in 3% nonfat, dried milk or 5% bovine serum albumin in TBS-Tween-20.

Techniques: Clinical Proteomics, Membrane

Journal: Nutrients

Article Title: Soluble Whey Protein Hydrolysate Ameliorates Muscle Atrophy Induced by Immobilization via Regulating the PI3K/Akt Pathway in C57BL/6 Mice

doi: 10.3390/nu12113362

Figure Lengend Snippet: The primer sequences.

Article Snippet: The primary antibodies were purchased from Cell Signaling (MA, USA) (p-PI3K (#4228), Akt (#9272), p-Akt (#9271), mTOR (#2972), p-mTOR (#2971), S6K1 (#9202), p-S6K1 (#9205), 4E-BP1 (#9452), p-4E-BP1 (#2855), FoxO3a (#12829), p-FoxO3a (#9465), and BNIP3 (#3769)), Abcam (Cambridge, UK) (PI3K (ab191606)), Sigma-Aldrich (MO, USA) (p62 (P0067)), GeneTex (CA, USA) (β-actin (GT5512) and GAPDH (GT239)), and Santa Cruz Biotechnology (CA, USA) (Atrogin-1 (sc-166806) and MurF1 (sc-398608)).

Techniques:

Journal: Molecular Metabolism

Article Title: Aryl hydrocarbon receptor maintains hepatic mitochondrial homeostasis in mice

doi: 10.1016/j.molmet.2023.101717

Figure Lengend Snippet: Direct regulation of Bnip3 transcription by AhR. (A) The mRNA level of Bnip3 and Ahr in the GEO dataset (GSE46495) during fed and fasted conditions. (n = 5, each). (B) Expression levels of Bnip3, Ahr, and Cyp1a1 in fed and fasted livers of WT or AhR KO mice (n = 3 or 4). Data were shown as box and whisker plots. Box, interquartile range (IQR); whiskers, min to the max; and horizontal line within box, median, ∗p < 0.05, ∗∗p < 0.01. (C) Pearson's correlation between Ahr and Bnip3 mRNA levels. (D) BNIP3 protein levels in fed and fasted livers of WT and AhR KO mice (upper) and IHC for BNIP3 in fasted livers (lower, left). Representative images were shown (n = 3 each). Scale bar, 100 μm. The quantification of BNIP3 expression (lower, right) was measured by Image J. (E) Increased BNIP3 expression by endogenous AhR ligand, kynurenine (Kyn) treatment (100 μM, 24 h) in mouse primary hepatocyte (left) and AML12 cells (middle and right). (F) AhR recruitment to the Bnip3 genomic locus in TCDD-treated liver (GSE97634). (G) ChIP-PCR analysis of AhR binding to Bnip3 genomic locus. (H) Reporter assays using the pGL3-basic vector containing WT ARE or Mutated ARE. HEK293 cells transfected with mock or AhR overexpression vector together with the reporter vector and treated Kyn for 24h. Results was normalized to WT control. (D), (E), (G) and (H), Data represented the mean ± SEM, ∗p < 0.05, ∗∗p < 0.01. (H), ## was compared to AhR overexpressed group.

Article Snippet: Antibodies against BNIP3 (nbp1-77683s), LC3A (NB100-2331), and LC3B (NB600-1384) were obtained from Novus Biologicals (Littleton, CO).

Techniques: Expressing, Whisker Assay, Binding Assay, Plasmid Preparation, Transfection, Over Expression, Control

Journal: Molecular Metabolism

Article Title: Aryl hydrocarbon receptor maintains hepatic mitochondrial homeostasis in mice

doi: 10.1016/j.molmet.2023.101717

Figure Lengend Snippet: Restored mitophagy by BNIP3 overexpression. (A) Changes of LC3A protein levels by Bnip3 overexpression in AhR knockdown cells. AML12 cells were co-transfected with siCon, siAhR, or siAhR with Bnip3-overexpressing plasmids. Band intensities represent values relative to siCon. (B) Changes of mitochondria-associated LC3A levels. Cytosolic and mitochondrial fractions were subjected to Western blotting. Cox IV was used for the control of mitochondrial fraction. (C) and (D) Visualization of mitophagy using mt-Keima assay and quantification. (E) Dual staining of mitochondria (Mitotracker) and autophagy marker (LC3A). Representative images are presented. Scale bar, 50 μm. (F) Quantification of colocalization of LC3A with mitotracker. (G) Mitochondrial superoxide levels measured by MitoSOX™ in AML12 cells transfected with siCon, siAhR, or siAhR with Bnip3 overexpression plasmids. (H) Mitoplate S-1 assay to measure mitochondrial substrate utilization. AML12 cells were transfected with siCon, siAhR, or siAhR with Bnip3-overexpressing plasmids for 48 h. The change in metabolic rate for each substrate/intermediate of mitochondrial/glycolytic pathways was shown in a heatmap (left) and change of substrate utilization in TCA cycle were shown as a bar graph (right). (A),(B), (D–H), Data represented the mean ± SEM, ∗p < 0.05, ∗∗p < 0.01. PPP; pentose phosphate pathway.

Article Snippet: Antibodies against BNIP3 (nbp1-77683s), LC3A (NB100-2331), and LC3B (NB600-1384) were obtained from Novus Biologicals (Littleton, CO).

Techniques: Over Expression, Knockdown, Transfection, Western Blot, Control, Staining, Marker

Journal: Hypoxia

Article Title:

Ascorbate modulates the hypoxic pathway by increasing intracellular activity of the HIF hydroxylases in renal cell carcinoma cells

doi: 10.2147/hp.s201643

Figure Lengend Snippet: Figure 2 Hypoxic induction of HIF pathway proteins in RCC cell lines. HIF pathway proteins were analysed by Western blotting following incubation of Caki-1, Caki-2 and 786-0 in decreasing oxygen tensions for 8 hrs. Bar charts show protein levels of HIF-1α (A, F, K), HIF-2α (B, G, L), GLUT1 (C, H, M), cyclin D1 (D, I, N) and BNIP3 (E, J, O) relative to a positive control (hypoxia-treated T24 cell lysate) which was loaded on each gel and referenced to β-actin. Data represent mean +SD from three independent experiments. Statistical significance was evaluated by One-way ANOVA with Dunnett’s Multiple Comparison Test; * p<0.05, ** p<0.01 *** p<0.001. Abbreviations: HIF-1α/2α, hypoxia-inducible factor-1α/2α; GLUT1, glucose transporter 1; BNIP3, Bcl2/adenovirus E1B 19 kDa interacting protein 3; ND, not detected.

Article Snippet: Membranes were incubated overnight at 4°C with primary antibodies against HIF-1α (1/800, BD Biosciences, SanJose, CA, USA, BD610958), hydroxy-HIF-1α (Pro564) (1/1000, Cell Signaling, Danvers, MA, USA, 3434), HIF-2α (1/400, R&D Systems, Minneapolis, MN, USA, AF2997), BNIP3 (1/1000, R&D Systems, AF4147), cyclin D1 (1/10,000, Abcam, Cambridge, UK, ab134175), GLUT1 (1/1000; Abcam, ab32551) or ß-actin (1/10,000, Sigma-Aldrich, Auckland, NZ, A5316), and for 1 hr at room temperature with secondary anti-goat, anti-mouse or anti-rabbit horseradish peroxidase-conjugated antibodies (1/5000, DAKO, Mulgrave, Australia) as appropriate.

Techniques: Western Blot, Incubation, Positive Control, Comparison

Journal: Hypoxia

Article Title:

Ascorbate modulates the hypoxic pathway by increasing intracellular activity of the HIF hydroxylases in renal cell carcinoma cells

doi: 10.2147/hp.s201643

Figure Lengend Snippet: Figure 3 Effect of ascorbate on HIF pathway protein levels at different oxygen tensions in Caki-1 cells. Cells were pre-loaded with increasing concentrations of ascorbate (0–1000 μM) for 16 hrs and then subjected to reduced oxygenation conditions. Shown are representative Western blots for HIF-1α, HIF-2α, GLUT1, cyclin D1 and BNIP3 from one of three independent experiments. β-actin was used as a loading control. Protein levels of HIF-1α and BNIP3 were decreased by ascorbate treatment under mild hypoxia (1–10% O2). Other proteins were not affected. Abbreviations: HIF-1α/2α, hypoxia-inducible factor-1α/2α; GLUT1, glucose transporter 1; BNIP3, Bcl2/adenovirus E1B 19 kDa interacting protein 3.

Article Snippet: Membranes were incubated overnight at 4°C with primary antibodies against HIF-1α (1/800, BD Biosciences, SanJose, CA, USA, BD610958), hydroxy-HIF-1α (Pro564) (1/1000, Cell Signaling, Danvers, MA, USA, 3434), HIF-2α (1/400, R&D Systems, Minneapolis, MN, USA, AF2997), BNIP3 (1/1000, R&D Systems, AF4147), cyclin D1 (1/10,000, Abcam, Cambridge, UK, ab134175), GLUT1 (1/1000; Abcam, ab32551) or ß-actin (1/10,000, Sigma-Aldrich, Auckland, NZ, A5316), and for 1 hr at room temperature with secondary anti-goat, anti-mouse or anti-rabbit horseradish peroxidase-conjugated antibodies (1/5000, DAKO, Mulgrave, Australia) as appropriate.

Techniques: Western Blot, Control

Journal: Hypoxia

Article Title:

Ascorbate modulates the hypoxic pathway by increasing intracellular activity of the HIF hydroxylases in renal cell carcinoma cells

doi: 10.2147/hp.s201643

Figure Lengend Snippet: Figure 5 Effect of ascorbate on HIF pathway protein levels at different oxygen tensions in 786-0 cells. Cells were pre-loaded with increasing concentrations of ascorbate (0–1000 μM) for 16 hrs and then subjected to reduced oxygenation conditions. Shown are representative Western blots for HIF-1α, HIF-2α, GLUT1, cyclin D1 and BNIP3 from one of three independent experiments. β-actin was used as a loading control. There was no consistent difference of protein levels after ascorbate treatment under any condition. Abbreviations: HIF-1α/2α, hypoxia-inducible factor-1α/2α; GLUT1, glucose transporter 1; BNIP3, Bcl2/adenovirus E1B 19 kDa interacting protein 3.

Article Snippet: Membranes were incubated overnight at 4°C with primary antibodies against HIF-1α (1/800, BD Biosciences, SanJose, CA, USA, BD610958), hydroxy-HIF-1α (Pro564) (1/1000, Cell Signaling, Danvers, MA, USA, 3434), HIF-2α (1/400, R&D Systems, Minneapolis, MN, USA, AF2997), BNIP3 (1/1000, R&D Systems, AF4147), cyclin D1 (1/10,000, Abcam, Cambridge, UK, ab134175), GLUT1 (1/1000; Abcam, ab32551) or ß-actin (1/10,000, Sigma-Aldrich, Auckland, NZ, A5316), and for 1 hr at room temperature with secondary anti-goat, anti-mouse or anti-rabbit horseradish peroxidase-conjugated antibodies (1/5000, DAKO, Mulgrave, Australia) as appropriate.

Techniques: Western Blot, Control

Journal: Hypoxia

Article Title:

Ascorbate modulates the hypoxic pathway by increasing intracellular activity of the HIF hydroxylases in renal cell carcinoma cells

doi: 10.2147/hp.s201643

Figure Lengend Snippet: Figure 4 Effect of ascorbate on HIF pathway protein levels at different oxygen tensions in Caki-2 cells. Cells were pre-loaded with increasing concentrations of ascorbate (0–1000 μM) for 16 hrs and then subjected to reduced oxygenation conditions. Shown are representative Western blots for HIF-1α, GLUT1, cyclin D1 and BNIP3 from one of three independent experiments. β-actin was used as a loading control. Protein levels of HIF-1α were increased after ascorbate treatment at 1% and 0.1% O2. Expression of all HIF targets was unchanged. Abbreviations: HIF-1α/2α, hypoxia-inducible factor-1α/2α; GLUT1, glucose transporter 1; BNIP3, Bcl2/adenovirus E1B 19 kDa interacting protein 3.

Article Snippet: Membranes were incubated overnight at 4°C with primary antibodies against HIF-1α (1/800, BD Biosciences, SanJose, CA, USA, BD610958), hydroxy-HIF-1α (Pro564) (1/1000, Cell Signaling, Danvers, MA, USA, 3434), HIF-2α (1/400, R&D Systems, Minneapolis, MN, USA, AF2997), BNIP3 (1/1000, R&D Systems, AF4147), cyclin D1 (1/10,000, Abcam, Cambridge, UK, ab134175), GLUT1 (1/1000; Abcam, ab32551) or ß-actin (1/10,000, Sigma-Aldrich, Auckland, NZ, A5316), and for 1 hr at room temperature with secondary anti-goat, anti-mouse or anti-rabbit horseradish peroxidase-conjugated antibodies (1/5000, DAKO, Mulgrave, Australia) as appropriate.

Techniques: Western Blot, Control, Expressing

Journal: Molecular cancer research : MCR

Article Title: Autophagy Induction Results in Enhanced Anoikis-resistance in Models of Peritoneal Disease

doi: 10.1158/1541-7786.MCR-16-0200-T

Figure Lengend Snippet: Directionality adjusted overlapping spheroid signature from across five cell lines

Article Snippet: BNIP3 (cat# TR316465), BCLN1 (TL314484V), and scrambled (SCR) shRNA were obtained from Origene.

Techniques: Translocation Assay, Binding Assay, Immunopeptidomics, Membrane, Ubiquitin Proteomics

Journal: Molecular cancer research : MCR

Article Title: Autophagy Induction Results in Enhanced Anoikis-resistance in Models of Peritoneal Disease

doi: 10.1158/1541-7786.MCR-16-0200-T

Figure Lengend Snippet: A. BNIP3 or consensus spheroid signature transcript levels correlate with survival in ovarian cancer patients. B. Representative IHC staining of ovarian cancer tissue showing high (4+) and low (1) staining of BNIP3. C. A significant difference in BNIP3 staining was noted between benign ovarian tissue and malignant tissue in the array, independent of primary or metastatic site. Level of BNIP3 in tumor tissue also correlated with survival in this cohort.

Article Snippet: BNIP3 (cat# TR316465), BCLN1 (TL314484V), and scrambled (SCR) shRNA were obtained from Origene.

Techniques: Immunohistochemistry, Staining

Journal: Molecular cancer research : MCR

Article Title: Autophagy Induction Results in Enhanced Anoikis-resistance in Models of Peritoneal Disease

doi: 10.1158/1541-7786.MCR-16-0200-T

Figure Lengend Snippet: A. BNIP3 increases in all cell lines in response to attachment independence. Beclin-1 and LC3b are increased in HEYA8 cells 12h after attachment loss and remain elevated throughout the spheroid time course. B. Spheroid morphology is changed in BECN1 knockdown compared to scramble shRNA. C. Pharmacologic perturbation of autophagy with rapamycin or hydroxychloroquine modulates the ability of HEYA8 and PANC1 cells to survive in attachment free conditions. D. Apoptosis increases with CQ treatment compared to rapamycin treated cells after loss of attachment.

Article Snippet: BNIP3 (cat# TR316465), BCLN1 (TL314484V), and scrambled (SCR) shRNA were obtained from Origene.

Techniques: Knockdown, shRNA

Journal: Molecular cancer research : MCR

Article Title: Autophagy Induction Results in Enhanced Anoikis-resistance in Models of Peritoneal Disease

doi: 10.1158/1541-7786.MCR-16-0200-T

Figure Lengend Snippet: When cell lose attachment, elevated BNIP3 allows dissociation of BECN1/BCL-2 complexes and allows for initiation of autophagy and inhibition of apoptosis to promote cell growth in tumorspheres. Autophagy modulators rapamycin and chloroquine can alter this response and cell survival in attachment free conditions.

Article Snippet: BNIP3 (cat# TR316465), BCLN1 (TL314484V), and scrambled (SCR) shRNA were obtained from Origene.

Techniques: Inhibition