Journal: The Journal of Biological Chemistry

Article Title: Requirement of hepatic pyruvate carboxylase during fasting, high fat, and ketogenic diet

doi: 10.1016/j.jbc.2022.102648

Figure Lengend Snippet: Selected transcripts from RNA-seq differential expression analysis of 24 h fasted male control and Pcx L−/− liver that were changed by more than 2-fold (n = 4/group)

Article Snippet: Membranes were blocked with 5% nonfat milk in Tris-buffered saline with Tween-20 (TBST) for 1 h and incubated overnight at 4 °C with primary antibodies at 1:1000 in 3% bovine serum albumin in TBST and all secondaries were diluted 1:5000 in 5% nonfat milk in TBST for 1 h. Antibodies used were as follows: pyruvate carboxylase (#16588-1-AP, Proteintech), HSC 70 (#sc-7298, Santa Cruz Biotechnology), pyruvate dehydrogenase (#2784 Cell Signaling), Citrate Synthase (#14309 Cell Signaling), Malate Dehydrogenase 2 (#11908 Cell Signaling), Mitochondrial Pyruvate Carrier 2 , Glutamine Oxaloacetate Transaminase 2 (HPA018139 Millipore Sigma), Carnitine Palmitoyltransferase 2 (PA5-12217 Thermo Fisher Scientific), Acyl-CoA Dehydrogenase Medium Chain (Genetex Irvine), Hydroxyacyl-CoA Dehydrogenase Trifunctional Multienzyme Complex Subunit Alpha (GTX101177 Genetex Irvine), Acyl-CoA Thioesterase 2 (15633-1-AP, Proteintech), Total Oxphos antibody cocktail (ab110411, Abcam), Acetylated-Lysine (#9441, Cell Signaling), mouse IgG-horseradish peroxidase (#7076, Cell Signaling), horseradish peroxidase–conjugated anti-rabbit (#NA934V Cytiva Life Sciences).

Techniques: Quantitative Proteomics, Control, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Requirement of hepatic pyruvate carboxylase during fasting, high fat, and ketogenic diet

doi: 10.1016/j.jbc.2022.102648

Figure Lengend Snippet: The loss of pyruvate carboxylase elicits mitochondrial protein hyperacetylation. A , Western blot of acetylated proteins from 9-week-old fed and 24 h fasted Pcx f/f and Pcx L−/− mice. B , Western blot of acetylated proteins from 9-week-old fed, 24 h fasted, 12 week high fat diet–fed, and 1 week ketogenic diet–fed Pcx f/f and Pcx L−/− mice. C , quantitative TMT acetyl-proteomics of 24 h fasted Pcx f/f and Pcx L−/− livers showing mitochondrial proteins (n = 5). D , quantitative TMT acetyl-proteomics of 24 h fasted Pcx f/f and Pcx L−/− livers showing all proteins (n = 5). E , Western blots of mitochondrial proteins from 9-week-old Pcx f/f and Pcx L−/− mice (n = 4). Pyruvate Carboxylase (Pcx), Pyruvate Dehydrogenase 1a (Pdh1a), Citrate Synthase (Cs), Malate Dehydrogenase 2 (Mdh2), Mitochondrial Pyruvate Carrier 2 (Mpc2), Glutamine Oxaloacetate Transaminase 2 (Got2), Carnitine Palmitoyltransferase 2 (Cpt2), Acyl-CoA Dehydrogenase Medium Chain (Acadm), Hydroxyacyl-CoA Dehydrogenase Trifunctional Multienzyme Complex Subunit Alpha (Hadha), Acyl-CoA Thioesterase 2 (Acot2), ATP synthase, H+ transporting, mitochondrial F1 complex, alpha subunit 1 (Atp5a), ubiquinol cytochrome c reductase core protein 2 (Uqcrc2), mitochondrially encoded cytochrome c oxidase I (Mtco1), succinate dehydrogenase complex, subunit B (Sdhb), NADH:ubiquinone oxidoreductase subunit B8 (Ndufb8).

Article Snippet: Membranes were blocked with 5% nonfat milk in Tris-buffered saline with Tween-20 (TBST) for 1 h and incubated overnight at 4 °C with primary antibodies at 1:1000 in 3% bovine serum albumin in TBST and all secondaries were diluted 1:5000 in 5% nonfat milk in TBST for 1 h. Antibodies used were as follows: pyruvate carboxylase (#16588-1-AP, Proteintech), HSC 70 (#sc-7298, Santa Cruz Biotechnology), pyruvate dehydrogenase (#2784 Cell Signaling), Citrate Synthase (#14309 Cell Signaling), Malate Dehydrogenase 2 (#11908 Cell Signaling), Mitochondrial Pyruvate Carrier 2 , Glutamine Oxaloacetate Transaminase 2 (HPA018139 Millipore Sigma), Carnitine Palmitoyltransferase 2 (PA5-12217 Thermo Fisher Scientific), Acyl-CoA Dehydrogenase Medium Chain (Genetex Irvine), Hydroxyacyl-CoA Dehydrogenase Trifunctional Multienzyme Complex Subunit Alpha (GTX101177 Genetex Irvine), Acyl-CoA Thioesterase 2 (15633-1-AP, Proteintech), Total Oxphos antibody cocktail (ab110411, Abcam), Acetylated-Lysine (#9441, Cell Signaling), mouse IgG-horseradish peroxidase (#7076, Cell Signaling), horseradish peroxidase–conjugated anti-rabbit (#NA934V Cytiva Life Sciences).

Techniques: Western Blot

Journal: Translational Cancer Research

Article Title: MicroRNA-497-5p negatively regulates the proliferation and cisplatin resistance of non-small cell lung cancer cells by targeting YAP1 and TEAD1

doi: 10.21037/tcr.2019.10.03

Figure Lengend Snippet: Primer sequences for quantitative real-time PCR

Article Snippet: YAP1 and TEAD1 expression pcDNA3.1 plasmids were purchased from Origene (Beijing, China).

Techniques: Sequencing

Journal: Translational Cancer Research

Article Title: MicroRNA-497-5p negatively regulates the proliferation and cisplatin resistance of non-small cell lung cancer cells by targeting YAP1 and TEAD1

doi: 10.21037/tcr.2019.10.03

Figure Lengend Snippet: YAP1 and TEAD1 are targeted by miR-497-5p. (A) Sequence comparison between miR-497-5p and the 3’-UTR region of YAP1 and TEAD1. A549 cells were transfected with miR-497-5p mimic or miR-497-5p inhibitor, the levels of YAP1 and TEAD1 are detected by qRT-PCR (B) and Western blot (C). (D) A549 and H1299 cells were co-transfected with miR-497-5p mimic and luciferase vectors carrying 3’-UTR of YAP1 or TEAD1, the luciferase activity was measured. (E) The levels of YAP1 and TEAD1 in clinical cancer specimens were evaluated by qRT-PCR. (F) The correlation between miR-497-5p and YAP1/TEAD1 was evaluated by Spearman’s rank correlation test. Data were mean ± SD and were representative of three independent experiments. *P<0.05; **P<0.01; ***P<0.001.

Article Snippet: YAP1 and TEAD1 expression pcDNA3.1 plasmids were purchased from Origene (Beijing, China).

Techniques: Sequencing, Comparison, Transfection, Quantitative RT-PCR, Western Blot, Luciferase, Activity Assay

Journal: Translational Cancer Research

Article Title: MicroRNA-497-5p negatively regulates the proliferation and cisplatin resistance of non-small cell lung cancer cells by targeting YAP1 and TEAD1

doi: 10.21037/tcr.2019.10.03

Figure Lengend Snippet: MiR-497-5p regulates the function of lung cancer cells via suppressing YAP1 and TEAD1. A549 and H1299 cells were co-transfected with miR-497-5p mimic and YAP1- or TEAD1-expressing plasmids, cell proliferation was measured by CCK-8 assay (A) and colony-formation assay (B). Cell cycle (C) and cell apoptosis (D) were evaluated by flow cytometry. The expression of YAP1 or TEAD1 was determined by qRT-PCR (E) and Western blot (F). Data were mean ± SD and were representative of three independent experiments. *P<0.05; **P<0.01.

Article Snippet: YAP1 and TEAD1 expression pcDNA3.1 plasmids were purchased from Origene (Beijing, China).

Techniques: Transfection, Expressing, CCK-8 Assay, Colony Assay, Flow Cytometry, Quantitative RT-PCR, Western Blot

Journal: Translational Cancer Research

Article Title: MicroRNA-497-5p negatively regulates the proliferation and cisplatin resistance of non-small cell lung cancer cells by targeting YAP1 and TEAD1

doi: 10.21037/tcr.2019.10.03

Figure Lengend Snippet: MiR-497-5p sensitizes lung cancer cells to cisplatin through the inhibition of YAP1 and TEAD1. (A) The expression miR-497-5p, YAP1 and TEAD1 in A549 cells and A549/DDP cells was evaluated by qRT-PCR. (B) The expression YAP1 and TEAD1 in A549 cells and A549/DDP cells was evaluated by western blot. (C,D) A549/DDP cells was transfected with miR-497-5p mimic followed by treatment with cisplatin, cell viability was evaluated by CCK-8 assay (C), cell apoptosis was measured by flow cytometry (D). (E) The levels of YAP1 and TEAD1 was evaluated in cisplatin-challenged A549/DDP cells by western blot. Data were mean ± SD and were representative of three independent experiments. *P<0.05; **P<0.01.

Article Snippet: YAP1 and TEAD1 expression pcDNA3.1 plasmids were purchased from Origene (Beijing, China).

Techniques: Inhibition, Expressing, Quantitative RT-PCR, Western Blot, Transfection, CCK-8 Assay, Flow Cytometry