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: Frontiers in Pharmacology

Article Title: Vitamin D Attenuates Ischemia/Reperfusion-Induced Cardiac Injury by Reducing Mitochondrial Fission and Mitophagy

doi: 10.3389/fphar.2020.604700

Figure Lengend Snippet: Vit D attenuated H/R-induced mitophagy in cardiomyocytes. (A) Cells were stained with acridine orange (AO) solution and observed under a fluorescence microscope to examine the changes in the acidic compartments of the cells. Scale bar = 50 μm. (B) Cells were stained with AO solution and analyzed by flow cytometry. AO green and red overlay fluorescence showed that H/R enhanced AO staining, whereas Vit D reduced it. The bar graph shows the percentages of cells undergoing autophagy (AO red positive and green positive) (n = 3). (C–E) Expression of BNIP3 and LC3BII/I was examined by Western blot (n = 5). (F–H) Measurement of the effects of MitoTEMPO on the expression of BNIP3 and LC3BII/I by Western blot (n = 5). (I) Measurement of the interaction of BNIP3 and LC3BII/I by co-immunoprecipitation. The indicated cultured cells were immunoprecipitated with anti-BNIP3 antibodies followed by immunoblotting with anti-LC3B antibodies. * p < 0.05 vs. control, † p < 0.05 vs. H/R.

Article Snippet: These membranes were probed overnight at 4 °C with the following primary antibodies: caspase 3, cytochrome c, p-Drp1, Mff, and LC3B, which were purchased from Cell Signaling (MA, USA), HIF-1α and Bax, which were purchased from GeneTex (Hsinchu city, Taiwan), Bcl-2, which was purchase from BD, and BNIP3, which was purchased from Aviva Systems Biology (CA, USA).

Techniques: Staining, Fluorescence, Microscopy, Flow Cytometry, Expressing, Western Blot, Immunoprecipitation, Cell Culture, Control

Journal: Frontiers in Pharmacology

Article Title: Vitamin D Attenuates Ischemia/Reperfusion-Induced Cardiac Injury by Reducing Mitochondrial Fission and Mitophagy

doi: 10.3389/fphar.2020.604700

Figure Lengend Snippet: Vit D reduced the translocation of mitochondrial fission-associated proteins (Drp1 and Mff) and mitophagy-associated proteins (BNIP3 and LC3B) in H/R-treated cardiomyocytes. (A) Mitochondrial translocation of Drp1, Mff, BNIP3, and LC3B in response to H/R treatment. H/R treatment induced a significant increase in Drp1, Mff, BNIP3 and LC3B translocation to mitochondria. Vit D treatment reduced this translocation. (B and C) Co-localization of COX IV (a mitochondrial marker) and Drp1 or LC3B was examined by dual immunofluorescence staining. The co-localization was increased in the H/R group compared to the control group, while Vit D treatment reduced this effect. Scale bar = 25 μm. (D) TEM showed that autophagic vacuoles (arrow) were present in the H/R-treated cells, and Vit D reduced the presence of autophagic vacuoles. The right panels are the magnification of the region enclosed in the red box. Scale bar = 500 nm. (E and F) The effect of Mdivi-1 (an inhibitor of mitochondrial fission) on the expression levels of BNIP3 and LC3BII/LC3BI ratio were analyzed by Western blot. LC3BII/LC3BI ratios reflect the autophagy activity (n = 3). (G and H) The effects of bafilomycin A1, an autophagy inhibitor, on the expression levels of BNIP3 and LC3BII/I ratio were analyzed by Western blot (n = 3). * p < 0.05 vs. control, † p < 0.05 vs. H/R.

Article Snippet: These membranes were probed overnight at 4 °C with the following primary antibodies: caspase 3, cytochrome c, p-Drp1, Mff, and LC3B, which were purchased from Cell Signaling (MA, USA), HIF-1α and Bax, which were purchased from GeneTex (Hsinchu city, Taiwan), Bcl-2, which was purchase from BD, and BNIP3, which was purchased from Aviva Systems Biology (CA, USA).

Techniques: Translocation Assay, Marker, Immunofluorescence, Staining, Control, Expressing, Western Blot, Activity Assay

Journal: Frontiers in Pharmacology

Article Title: Vitamin D Attenuates Ischemia/Reperfusion-Induced Cardiac Injury by Reducing Mitochondrial Fission and Mitophagy

doi: 10.3389/fphar.2020.604700

Figure Lengend Snippet: Vit D reduced I/R-induced cardiac injury. (A) Transverse sections of cardiac morphology were observed by haematoxylin and eosin staining. I/R induced more inflammatory cell infiltration into cardiac tissue than the control, while Vit D ameliorated this inflammatory infiltration. Scale bar = 600 μm or 100 μm, as indicated in the panel. (B) The production of superoxide anions was assessed by DHE staining. Scale bar = 100 μm. (C) Representative ultrastructural images were observed by TEM. Mitophagy was present in the I/R-treated cardiomyocytes. The control and Vit D treatment groups exhibited intact mitochondrial structures with well-arranged cristae. Scale bar = 500 nm. (D) Myocardial apoptosis was identified and quantified by TUNEL assay (TUNEL: brown; nuclei: blue; scale bar = 100 μm). (E) The protein levels of HIF-1α, Drp1, Mff, BNIP3, LC3B, and cleaved caspase three were determined by Western blot. Vit D treatment significantly reduced the levels of these proteins in the I/R-treated mice. (F) The effect of bafilomyocin A1, an autophagy inhibitor, on the levels of BNIP3 and LC3BII/I expression in I/R-treated mice (n = 3). * p < 0.05 vs. control, † p < 0.05 vs. I/R.

Article Snippet: These membranes were probed overnight at 4 °C with the following primary antibodies: caspase 3, cytochrome c, p-Drp1, Mff, and LC3B, which were purchased from Cell Signaling (MA, USA), HIF-1α and Bax, which were purchased from GeneTex (Hsinchu city, Taiwan), Bcl-2, which was purchase from BD, and BNIP3, which was purchased from Aviva Systems Biology (CA, USA).

Techniques: Staining, Control, TUNEL Assay, Western Blot, Expressing

Journal: Physiological reports

Article Title: High-fat diet increases electron transfer flavoprotein synthesis and lipid respiration in skeletal muscle during exercise training in female mice.

doi: 10.14814/phy2.15840

Figure Lengend Snippet: FIGURE 8 Twelve weeks of high-fat diet induced mitochondrial remodeling through fission, fusion, and BNIP3-dependent autophagy and mitophagy mechanisms. (a) markers of mitochondrial dynamics (fission and fusion) and (b) representative blots. (c) whole-cell autophagy and (d) representative blots. (e) markers of mitophagy in mitochondrial subfractions (subsarcolemmal [SSM] and intermyofibrillar [IMFM]) and (f) representative blots. Whole-cell lysates are from quadriceps and isolated mitochondria are from gastrocnemius. Data displayed as mean ± SD. Effects of diet and exercise were evaluated using two-way ANOVA. Images on representative blots come from separate membranes and channels.

Article Snippet: Secondary antibodies were diluted in 5% BSA-TBST or 5% nonfat dry milk-TBST and membranes incubated at room temperature for 1 h. Primary antibodies were diluted 1:1000 and purchased from Abcam (Cambridge, United Kingdom) for OXPHOS cocktail (110413), ETFα (110316), ETFβ (240593), Trimethylated ETFβ (76118), ETFDH (103910), CPT1 (134988), PINK1 (23707), Mitochondrial fission factor (MFF) (81127); Cell signaling (Danvers, MA) for LC3 (12741), p62 (39749), Parkin (4211), and BNIP3 (3769), OPA1 (80471), mitofusion 2 (MFN2) (9482), Abcam, DRP1 (14647); Other primary antibodies included METTL20 (87995, Novus Biologicals, Littleton, CO), HADH (PA5-28203, Thermo Fisher Scientific, Waltham, MA), and BCL2 (7382, Santa Cruz Biotechnology, Dalla, TX).

Techniques: Isolation