Journal: Cell Reports Medicine

Article Title: Urolithin A improves muscle strength, exercise performance, and biomarkers of mitochondrial health in a randomized trial in middle-aged adults

doi: 10.1016/j.xcrm.2022.100633

Figure Lengend Snippet: Urolithin A confers a proteomic signature of improved mitochondrial metabolism and mitophagy in human skeletal muscle (A) Venn diagram summarizing pathway enrichment analysis results from proteomics data in vastus lateralis skeletal muscle. Data represent upregulated pathways with an adjusted p value <0.1 in subjects treated with placebo or with UA at 500 and 1,000 mg for 4 months compared with baseline. (B and C) Dot plots showing top enriched pathways (WikiPathways 2019 Human), ranked by protein ratio, in the UA 500- (B) and UA 1,000 mg (C) groups from (A). Dot color and size indicate adjusted p value and protein count, respectively. Significant pathways for the placebo group were filtered out to identify treatment-specific pathways. (D) Western blot analysis of protein lysates from vastus lateralis skeletal-muscle biopsies in subjects treated as above. For each subject, both baseline (Pre) and end-of-the-treatment (Post) samples were run, and membranes were probed for phospho-Parkin, total Parkin, and the mitochondrial proteins ATP5A (belonging to the OXPHOS complex V), UQCRC2 (complex IV), SDHB (complex II), and NDUFB8 (complex I). Tubulin and VDAC were included as markers of total and mitochondrial protein abundance, respectively. Dashed line separates samples from individual subjects. (n = 6 Pre and Post, biologically independent samples). (E) Quantification of phospho-Parkin over Tubulin protein intensity from western blots (WBs) in (D) (n = 6). Two-sided, paired t-test. (F) Quantification of NDUFB8 (left) and SDHB (right) protein intensity, normalized over VDAC from WBs in (D) (n = 6). ∗p < 0.05; ∗∗p < 0.01; two-sided, paired t test.

Article Snippet: The following primary antibodies were incubated overnight diluted in blocking buffer: UBE2N (SantaCruz, sc-376470, 1:3000), Tubulin (Proteintech, 10004185, 1:3000), phospho-Parkin S65 (Cell Signaling, #36866, 1:1000), Parkin (Cell Signaling, #4211, 1:1000), OXPHOS Antibody Cocktail (Abcam, ab110413, 1:2000), VDAC (Proteintech, 10866-1-AP, 1:1000).

Techniques: Western Blot, Quantitative Proteomics

Journal: Cell Reports Medicine

Article Title: Urolithin A improves muscle strength, exercise performance, and biomarkers of mitochondrial health in a randomized trial in middle-aged adults

doi: 10.1016/j.xcrm.2022.100633

Figure Lengend Snippet:

Article Snippet: The following primary antibodies were incubated overnight diluted in blocking buffer: UBE2N (SantaCruz, sc-376470, 1:3000), Tubulin (Proteintech, 10004185, 1:3000), phospho-Parkin S65 (Cell Signaling, #36866, 1:1000), Parkin (Cell Signaling, #4211, 1:1000), OXPHOS Antibody Cocktail (Abcam, ab110413, 1:2000), VDAC (Proteintech, 10866-1-AP, 1:1000).

Techniques: Recombinant, RNA Extraction, Software, Protease Inhibitor

Journal: Molecular nutrition & food research

Article Title: Lack of β, β-carotene -9’, 10’-oxygenase 2 leads to hepatic mitochondrial dysfunction and cellular oxidative stress in mice

doi: 10.1002/mnfr.201600576

Figure Lengend Snippet: Functional categorization of proteins differentially expressed in hepatic mitochondria of BCO2 knockout (KO) vs. 129S6 wild type (WT) mice identified by spectrum counting. Values are means, n=3 mice/group with 4 technical replicates/mouse.

Article Snippet: Antibodies against fatty acid synthase (FASN) (catalog # 10624-2-Ap), ATP synthase α subunit 1 (ATP5A1) (catalog # 14676-1-AP), citrate synthase (catalog # 16131-1-AP), hydroxyacyl-coenzyme A dehydrogenase (HADH) (catalog # 19828-1-AP), succinate dehydrogenase α (SDHA) (catalog # , 14865-1-AP), and nicotinamide nucleotide transhydrogenase (NNT) (catalog # 13442-2-AP) were purchased from ProteinTech Group (Chicago, IL, USA); Antibodies against lysosome-associated membrane protein 1 (LAMP1) (catalog # sc-20011), catalase (catalog # sc-50508), NNT (catalog # sc-163154), glutathione reductase (GSR) (catalog # sc-133245) were purchased from Santa Cruz Biotech (Dallas, TX, USA); peroxisome proliferator-activated receptor α (PPARα) antibody (catalog # 101710) was purchased from Cayman (Ann Arbor, MI, USA); Antibodies against β-actin (catalog # 4967), phosphor-Thr172-AMP-activated protein kinase α (pT172-AMPKα) (catalog # 2535), and AMPKα (catalog # 2603) were purchased from Cell Signaling Technology (Danvers, MA, USA); voltage-dependent anion-selective channel protein 1 (VDAC1) antibody (catalog # PA1780) was purchased from Boster Biosciences (Pleasanton, CA, USA).

Techniques: Functional Assay, Knock-Out, Membrane

Journal: Experimental and Therapeutic Medicine

Article Title: Penehyclidine hydrochloride post-conditioning reduces ischemia/reperfusion-induced cardiomyocyte apoptosis in rats

doi: 10.3892/etm.2017.5089

Figure Lengend Snippet: Effect of PHC on the expression of apoptosis-related proteins. Following 3 h reperfusion, the expression of apoptosis-related proteins was analyzed using western blotting. Band densities were normalized to β-actin or COX IV. (A) Bax protein expression. (B) Bcl-2 protein expression. (C) VDAC1 protein expression. (D) Cytosol cyt- c protein expression. (E) Cleaved caspase-3 protein expression. Data are expressed as mean ± standard deviation (n=6). ## P<0.01 vs. sham; **P<0.01 vs. I/R. ns, not significant (P>0.05); I/R, ischemia/reperfusion; PHC, penehyclidine hydrochloride; COX IV, cytochrome c oxidase subunit 4; VDAC1, voltage dependent-selective anion channel protein 1; cyt- c , cytochrome- c .

Article Snippet: Following blocking with 5% non-fat milk for 1 h and washing with TTBS (150 mM NaCl, 20 mM Tris, 0.1% Tween-20, pH 7.6) for 5 min, membranes were incubated with specific Bcl-2 antibody (1:500; cat. no/WL01556), Bax antibody (1:500; cat. no. WL01637; both from Wanleibio Co., Ltd.), VDAC1 antibody (1:500, cat. no. bs-1461R; Bioss, Beijing, China), β-actin antibody (1:1,000, WL01845), COX IV (1:500, WL01794) or cyt- c antibody (1:500, WL01571) (all from Wanleibio Co., Ltd.) overnight at 4°C and then with goat anti-rabbit immunoglobulin G conjugated to horseradish peroxidase (1:5,000, cat. no. WLA023a; Wanleibio Co., Ltd.) for 45 min at 37°C.

Techniques: Expressing, Western Blot, Standard Deviation

Journal: Molecular Therapy. Nucleic Acids

Article Title: Overexpression of lncRNA EPB41L4A-AS1 Induces Metabolic Reprogramming in Trophoblast Cells and Placenta Tissue of Miscarriage

doi: 10.1016/j.omtn.2019.09.017

Figure Lengend Snippet: EPB41L4A-AS1 Overexpression Mediates Metabolic Reprogramming (A) Seahorse XFp assays measured the oxygen consumption rate (OCR) in EPB41L4A-AS1 overexpression in HTR8 cells. (B) Basal respiration, ATP production, maximal respiration, spare respiratory capacity, proton leak, and non-mitochondrial respiration were increased in the EPB41L4A-AS1 overexpression group, and the difference reached statistical significance for maximal respiration (p < 0.05). (C) Seahorse XFp assays measured the oxygen consumption rate (OCR) in EPB41L4A-AS1 knockdown in HTR8 cells. Black arrows indicate the time points of cell treatment with different chemicals. The result showed that OCRs were significantly promoted in the EPB41L4A-AS1 overexpression group (p < 0.05). (D) Basal respiration, ATP production, maximal respiration, spare respiratory capacity, proton leak, and non-mitochondrial respiration were decreased in the EPB41L4A-AS1 siRNA group, and the difference reached statistical significance for maximal respiration (p < 0.05). (E) PGC-1α, VDAC1, and PDK4 expression was higher in HTR8 cells with EPB41L4A-AS1 overexpression but lower in HTR8 cells with EPB41L4A-AS1 knockdown. (F) Intracellular fatty acid oxidation was raised after EPB41L4A-AS1 overexpression, but there was no significant difference after EPB41L4A-AS1 knockdown in HTR8 cells. (G) The intracellular carnitine concentration was elevated after EPB41L4A-AS1 overexpression but decreased after EPB41L4A-AS1 knockdown in HTR8 cells. (H) The concentration of total fatty acid was detected by colorimetric assays in HTR8 cells after EPB41L4A-AS1 knockdown or overexpression with 24 h and 48 h. The fatty acid concentration was significantly decreased in the EPB41L4A-AS1 group both in cultured with 24 h and 48 h. (I) The intracellular fatty acid concentration was reduced in RM placental tissue. (J) The expression of oxidation-related enzymes (PDK4, VDAC1, and PGC-1α) were increased in the EPB41L4A-AS1 overexpression group but decreased in the EPB41L4A-AS1 knockdown group. All data are represented as means ± SD; *p < 0.05, **p < 0.01.

Article Snippet: They were then blocked with 5% BSA in TBST buffer within 30 min and incubated for 1–2 h with the following primary antibodies: TIGA1 (Proteintech, 24698-1-AP), β-actin (Proteintech, 60008), VDAC1 (Novus Biologicals, NBP100-695), PGC-1α (Cell Signaling Technology, 5536), LDHA (Cell Signaling Technology, 3582), HIF-1α (Abcam, ab1), HK2 (Cell Signaling Technology, 2867), FASN (Cell Signaling Technology, 3180), PDK4 (Novus Biologicals, NBP1-54723), and CPT1 (Cell Signaling Technology, 12252).

Techniques: Over Expression, Knockdown, Expressing, Concentration Assay, Cell Culture

Journal: Molecular Therapy. Nucleic Acids

Article Title: Overexpression of lncRNA EPB41L4A-AS1 Induces Metabolic Reprogramming in Trophoblast Cells and Placenta Tissue of Miscarriage

doi: 10.1016/j.omtn.2019.09.017

Figure Lengend Snippet: EPB41L4A-AS1 Is a lncRNA that Performs Biological Functions (A) and (B) HIF-1α and VDAC1 gene expression was determined by qRT-PCR transfection with siEPB41L4A-AS1, the EPB41L4A-AS1 plasmid, or the EPB41L4A-AS1 ATG mutation plasmid. HIF-1α expression was increased in the EPB41L4A-AS1 knockdown group but decreased in the EPB41L4A-AS1 overexpression and EPB41L4A-AS1 ATG mutation groups; VDAC1 showed the opposite results. (C) Expression levels of VDAC1 mRNA and EPB41L4A-AS1 were positively correlated in early RM placental tissue (N = 73). (D) Expression levels of HIF-1α mRNA and EPB41L4A-AS1 were negatively correlated in early RM placental tissue (N = 73). (E) HIF-1α protein expression was higher but that of VDAC1 was lower in the EPB41L4A-AS knockdown group. (F) HIF-1α protein expression was downregulated in the EPB41L4A-AS1 and Mut-TIGA1 groups, but VDAC1 protein expression was upregulated in HTR8 cells. (G) HIF-1α protein expression was downregulated but VDAC1 was upregulated in RM placental tissue. (H) and (I) HIF-1α and VDAC1 protein expression was subjected to quantitative analysis by immunohistochemistry staining of RM placental tissue, and the difference reached statistical significance in the study group. (J) Cell numbers were calculated after EPB41L4A-AS1 overexpression or EPB41L4A-AS1 knockdown with different culture times (24, 48, 72, and 96 h). The cell numbers were reduced significantly in the EPB41L4A-AS1 group after culture for 72 h. (K) The expression of CASP3 and cleaved CASP3 was detected after EPB41L4A-AS1 overexpression with different culture times (24, 48, 72, and 96 h). Cell apoptosis was increased in the EPB41L4A-AS1 overexpression group after incubation for 72 h. (L) Cell apoptosis was raised by flow cytometer detection in the EPB41L4A-AS1 overexpression group. (M) The expression of CASP3 and cleaved CASP3 was increased after transfection with EPB41L4A-AS1, and this was aggravated by co-transfection with the VDAC1 plasmid but rescued after co-transfection with the HIF-1α plasmid with a different culture time. (N) The cell numbers were decreased after transfection with the EPB41L4A-AS1 and EPB41L4A-AS1+VDAC1 plasmids but rescued by the HIF-1α plasmid in the EPB41L4A-AS1 overexpression group within different culture times (24, 48, 72, and 96 h). Data are represented as means ± SD; *p < 0.05, **p < 0.01.

Article Snippet: They were then blocked with 5% BSA in TBST buffer within 30 min and incubated for 1–2 h with the following primary antibodies: TIGA1 (Proteintech, 24698-1-AP), β-actin (Proteintech, 60008), VDAC1 (Novus Biologicals, NBP100-695), PGC-1α (Cell Signaling Technology, 5536), LDHA (Cell Signaling Technology, 3582), HIF-1α (Abcam, ab1), HK2 (Cell Signaling Technology, 2867), FASN (Cell Signaling Technology, 3180), PDK4 (Novus Biologicals, NBP1-54723), and CPT1 (Cell Signaling Technology, 12252).

Techniques: Gene Expression, Quantitative RT-PCR, Transfection, Plasmid Preparation, Mutagenesis, Expressing, Knockdown, Over Expression, Immunohistochemistry, Staining, Incubation, Flow Cytometry, Cotransfection

Journal: Molecular Therapy. Nucleic Acids

Article Title: Overexpression of lncRNA EPB41L4A-AS1 Induces Metabolic Reprogramming in Trophoblast Cells and Placenta Tissue of Miscarriage

doi: 10.1016/j.omtn.2019.09.017

Figure Lengend Snippet: EPB41L4A-AS1 Recruits SET1A to Enhance the H3K4me3 Level in the VDAC1 Promoter and Regulates HIF-1α Expression via HIF1A-AS1 (A) RIP assays were performed using H3K4me3, H3K9me3, and H3K27Ac antibodies, and enrichment of EPB41L4A-AS1 was detected by qRT-PCR in HTR8 cells. EPB41L4A-AS1 was significantly enriched after incubation with H3K4me3 antibody (p < 0.05). (B) EPB41L4A-AS1 was enriched in JEG3 and BeWo cells after incubation with H3K4me3 antibody (p < 0.05). (C) The interaction of full-length EPB41L4A-AS1, the EPB41L4A-AS1 ATG mutation, and coding sequece region RNA with SET1A and H3K4me3 was evaluated by RNA pulldown assay. Full-length EPB41L4A-AS1 was combined with H3K4me3 antibody and the SET1A complex. (D) qRT-PCR and western blot assays showed that VDAC1 mRNA and protein expression was downregulated after SET1A knockdown in HTR8 cells. (E) The enrichment of H3K4me3 in the promoter regions of VDAC1 were increased via ChIP assays after transfection with the EPB41L4A-AS1 plasmid and the EPB41L4A-AS1 ATG mutation plasmid in HTR8 cells but decreased in the EPB41L4A-AS1 knockdown group. (F) HIF-1α gene and protein expression was elevated after SET1A knockdown in HTR8 cells, which was examined by qRT-PCR and western blot assays. (G) HIF-1α was increased in the EPB41l4A-AS1 knockdown group and decreased in the EPB41L4A-AS1 and EPB41L4A-AS1 ATG mutation groups in HTR8 cells. HIF1A-AS1 showed the opposite result. (H) Structure chart of HIF-1α and HIF1A-AS1.The expression of HIF-1α was increased in SET1A siRNA1 but decreased in SET1A siRNA2. (I) The enrichment of HIF1A-AS1 was detected by qRT-PCR in HTR8/BeWo/JEG3 cells after treatment with H3K4me3, H3K9me3, and H3K27Ac antibodies using RIP assays. HIF1A-AS1 was significantly enriched after incubation with H3K4me3 antibody, and similar results were found for BeWo and JEG3 cells. (J) The enrichment of HIF1A-AS1 was identified via ChIP assays after transfection with siEPB41l4A-AS1, EPB41L4A-AS1, or EPB41L4A-AS1 ATG mutation in HTR8 cells. HIF1A-AS1 was increased in the EPB41L4A-AS1 and EPB41L4A-AS1 ATG mutation groups and decreased in the EPB41L4A-AS1 knockdown group. (K) Schematic model of mediation by EPB41L4AAS1 in shifting the metabolic reprogramming of early RM. Data are represented as means ± SD; *p < 0.05, **p < 0.01, unpaired two-tailed Student’s t test.

Article Snippet: They were then blocked with 5% BSA in TBST buffer within 30 min and incubated for 1–2 h with the following primary antibodies: TIGA1 (Proteintech, 24698-1-AP), β-actin (Proteintech, 60008), VDAC1 (Novus Biologicals, NBP100-695), PGC-1α (Cell Signaling Technology, 5536), LDHA (Cell Signaling Technology, 3582), HIF-1α (Abcam, ab1), HK2 (Cell Signaling Technology, 2867), FASN (Cell Signaling Technology, 3180), PDK4 (Novus Biologicals, NBP1-54723), and CPT1 (Cell Signaling Technology, 12252).

Techniques: Expressing, Quantitative RT-PCR, Incubation, Mutagenesis, Western Blot, Knockdown, Transfection, Plasmid Preparation, Two Tailed Test

Journal: Cancer Reports

Article Title: A novel polyethylene glycol ( PEG )‐drug conjugate of Venetoclax, a Bcl‐2 inhibitor, for treatment of acute myeloid leukemia ( AML )

doi: 10.1002/cnr2.1485

Figure Lengend Snippet: Bax expression levels and cytochrome c concentrations in the cytoplasm or mitochondria following treatment with either free VTX or PEG‐VTX in vitro. MV4‐11 cells were seeded into a T25 flask (2.5 × 10 6 cells/flask) and were exposed to either free VTX or PEG‐VTX (0.01, 0.1, or 1 μM as an API VTX concentration). After 5 h of incubation, the cytoplasm and mitochondria fractions were collected. (A) The expression levels of Bax and β‐Actin in cytoplasm fraction and those of Bax and VDAC1/2 in mitochondria fraction were determined using a Wes system. The lower graphs show the relative signal intensity of Bax corrected by that of β‐Actin for cytoplasm fraction or that corrected by VDAX1/2 for mitochondria fraction. The data represent the mean ± SD ( n = 3, ** p < .01, *** p < .001 vs. control). (B) The concentrations of cytochrome c in these fractions was measured via ELISA, and corrected using these protein concentrations. The data represent a typical result from three independent experiments

Article Snippet: The expression of VDAC1/2, as a loading control for mitochondrial fraction, was detected using an anti‐VDAC1/2 primary antibody (10866‐1‐AP; Proteintech) at a dilution of 1:100.

Techniques: Expressing, In Vitro, Concentration Assay, Incubation, Enzyme-linked Immunosorbent Assay