Journal: Frontiers in Cell and Developmental Biology

Article Title: Flavin-Containing Monooxygenases Are Conserved Regulators of Stress Resistance and Metabolism

doi: 10.3389/fcell.2021.630188

Figure Lengend Snippet: C. elegans FMO-2 and mammalian FMOs improve stress resistance to oxidative stress. (A) Amino acid sequence alignment of FMOs across species with the regions containing the eight essential residues denoted as red arrowheads in the catalytic active site among reconstructed ancestral mammalian FMO5, C. elegans FMO-2, and mouse FMO1–5. (B) Wild-Type and FMO-2 overexpressing (FMO-2 OE) worm survival curves on paraquat stress. Each strain was placed on NGM (Nematode Growth Medium) containing 5 mM paraquat from the fourth larvae stage (L4) at Day 0. Survival was quantified every day until all worms were dead. The difference between the survival curves is denoted with the log-rank test p -value. (C) FMO1–5 protein levels in FMO1–5 OE and control cells of both HEK293A and HepG2. (D) FMO5 OE and control cell survival curves on paraquat stress in HEK293A and HepG2. HEK293A cells or HepG2 cells stably expressing FMO5 or empty vector pDEST were subjected to indicated increasing doses of paraquat. (E) LD 50 values of FMO1–5 OE cells compared to the control cells on paraquat stress in HEK293A and HepG2. HEK293A cells or HepG2 cells stably expressing FMO1–5 or empty vector pDEST were subjected to indicated increasing doses of paraquat. (F) FMO5 OE and control cell survival curves on cadmium stress in HEK 293A and HepG2. HEK293A cells or HepG2 cells stably expressing FMO5 or empty vector pDEST were subjected to indicated increasing doses of cadmium. (G) LD 50 values of FMO-OE cells compared to the control cells on cadmium stress in HEK293A and HepG2. HEK293A cells or HepG2 cells stably expressing FMO or empty vector pDEST were subjected to indicated increasing doses of paraquat. Data represent mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Mouse FMO1 (Accession No. U87456 ), FMO2 (Accession No. AF184981 ), and FMO3 (Accession No. NM_008030 ) were cloned from mouse liver cDNA (ZYAGEN) and inserted into Gateway TM pcDNA TM -DEST47 Vector (Invitrogen 12281010).

Techniques: Sequencing, Stable Transfection, Expressing, Plasmid Preparation

Journal: Frontiers in Cell and Developmental Biology

Article Title: Flavin-Containing Monooxygenases Are Conserved Regulators of Stress Resistance and Metabolism

doi: 10.3389/fcell.2021.630188

Figure Lengend Snippet: FMOs improve stress resistance to broader stressors in mammalian cells. (A,B) FMO1–5 OE and control cell survival curves on arsenite stress in HEK293A and HepG2. HEK293A cells (A) or HepG2 cells (B) stably expressing FMO1–5 or empty vector pDEST were subjected to indicated increasing doses of arsenite. (C) FMO5 OE and control cell survival curves on UV-radiation in HEK 293A and HepG2. HEK293A cells or HepG2 cells stably expressing FMO5 or empty vector pDEST were subjected to indicated increasing energies of UV-radiation. (D) LD 50 values of FMO1–5 OE cells compared to the control cells on UV-radiation in HEK293A and HepG2. HEK293A cells or HepG2 cells stably expressing FMO1–5 or empty vector pDEST were subjected to indicated increasing energies of UV-radiation. Data represent mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Mouse FMO1 (Accession No. U87456 ), FMO2 (Accession No. AF184981 ), and FMO3 (Accession No. NM_008030 ) were cloned from mouse liver cDNA (ZYAGEN) and inserted into Gateway TM pcDNA TM -DEST47 Vector (Invitrogen 12281010).

Techniques: Stable Transfection, Expressing, Plasmid Preparation

Journal: Frontiers in Cell and Developmental Biology

Article Title: Flavin-Containing Monooxygenases Are Conserved Regulators of Stress Resistance and Metabolism

doi: 10.3389/fcell.2021.630188

Figure Lengend Snippet: JNK kinase activity is increased in FMO-overexpressing cells under cadmium-induced oxidative stress. (A) SAPK levels and phosphorylation including JNK, p38, and ERK after 10 μM cadmium treatment of indicated time. HEK293A cells were treated with cadmium, and the SAPK activities were measured with antibodies against phosphorylated SAPKs. (B) SAPKs levels and phosphorylation including JNK, p38, and ERK in FMO1–5 OE HEK293A cells and empty vector control cells after 10 μM Cadmium treatment for 4 h. Quantitation of the phosphorylated JNK (C) , p38 (D) , and ERK (E) after 10 μM Cadmium treatment for 4 h in FMO 1–5 OE HEK293A cells [lane 8–12 in (B) ] compared to empty vector control HEK293A cells [lane 7 in (B) ]. (F) SAPK levels and phosphorylation including JNK, p38, and ERK in FMO1–5 OE HepG2 cells and empty vector control cells after 10 μM Cadmium treatment for 4 h. Quantitation of the phosphorylated JNK (G) , p38 (H) , and ERK (I) after 10 μM Cadmium treatment for 4 h in FMO1–5 OE HepG2 cells [lane 8–12 in (F) ] compared to empty vector control HepG2 cells [lane 7 in (F) ]. Western blot band intensities were quantified by Image J. The phosphorylated bands were normalized to the corresponding unphosphorylated bands of each SAPK, and then were compared to the empty vector pDEST control as fold changes. Data represent mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Mouse FMO1 (Accession No. U87456 ), FMO2 (Accession No. AF184981 ), and FMO3 (Accession No. NM_008030 ) were cloned from mouse liver cDNA (ZYAGEN) and inserted into Gateway TM pcDNA TM -DEST47 Vector (Invitrogen 12281010).

Techniques: Activity Assay, Plasmid Preparation, Quantitation Assay, Western Blot

Journal: Frontiers in Cell and Developmental Biology

Article Title: Flavin-Containing Monooxygenases Are Conserved Regulators of Stress Resistance and Metabolism

doi: 10.3389/fcell.2021.630188

Figure Lengend Snippet: FMO expression increases mitochondrial respiration. (A) Mitochondrial respiration measured by OCR (oxygen consumption rate) in FMO1–5 OE HEK293A cells and empty vector control cells. Mitochondrial respiration chain complex inhibitors Oligomycin, Carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP), and rotenone/antimycin A were injected stepwise as indicated. As denoted in the first panel, basal respiration, ATP production, maximal respiration, and spare respiration can be calculated by the OCR level changes in response to the inhibitor injections. (B) Basal respiration, ATP production, maximal respiration, and spare capacity in FMO1–5 OE HEK293A cells and empty vector control cells. (C) Mitochondrial respiration in FMO1–5 OE HepG2 cells. (D) Basal respiration, ATP production, maximal respiration, and spare capacity in FMO1–5 OE HepG2 cells. Data represent mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Mouse FMO1 (Accession No. U87456 ), FMO2 (Accession No. AF184981 ), and FMO3 (Accession No. NM_008030 ) were cloned from mouse liver cDNA (ZYAGEN) and inserted into Gateway TM pcDNA TM -DEST47 Vector (Invitrogen 12281010).

Techniques: Expressing, Plasmid Preparation, Injection

Journal: Frontiers in Cell and Developmental Biology

Article Title: Flavin-Containing Monooxygenases Are Conserved Regulators of Stress Resistance and Metabolism

doi: 10.3389/fcell.2021.630188

Figure Lengend Snippet: FMO overexpression decreases overall glycolytic activity. (A) Overall glycolytic activity measured by ECAR (extracellular acidification rates) in HEK293A FMO1–5 OE cells and empty vector control cells. Sequential injections of glucose, oligomycin, and 2-DG were applied over time as indicated. As denoted in the first panel, glycolysis, glycolytic capacity, and glycolytic reserve can be calculated by the ECAR level changes in response to individual injections. (B) Glycolysis, glycolytic capacity, and glycolytic reserve in HEK293A FMO1–5 OE cells and empty vector control cells. (C) Overall glycolytic activity in FMO1–5 OE HepG2 cells. (D) Glycolysis, glycolytic capacity, and glycolytic reserve in FMO1–5 OE HepG2 cells and empty vector control cells. Data represent mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Mouse FMO1 (Accession No. U87456 ), FMO2 (Accession No. AF184981 ), and FMO3 (Accession No. NM_008030 ) were cloned from mouse liver cDNA (ZYAGEN) and inserted into Gateway TM pcDNA TM -DEST47 Vector (Invitrogen 12281010).

Techniques: Over Expression, Activity Assay, Plasmid Preparation

Journal: Frontiers in Cell and Developmental Biology

Article Title: Flavin-Containing Monooxygenases Are Conserved Regulators of Stress Resistance and Metabolism

doi: 10.3389/fcell.2021.630188

Figure Lengend Snippet: FMOs regulate amino acid and energetic metabolic pathways. Untargeted metabolomics in FMO1–5 OE cells ( A , see also – ) compared to empty vector control cells, and the significantly regulated metabolic pathways that are enriched. The abundance analyses of metabolites in central carbon metabolism in FMO1–5 OE cells compared to empty vector control cells ( B,C , see also – ). (A) Metabolic pathways regulated by FMO1 are plotted by the enrichment factor (obtained by dividing “significant hits” by “expected hits” for each pathway) on the x -axis and –log of the p -value on the y -axis. Red indicates significantly changed pathways with p < 0.001. Shared significantly regulated metabolic pathways by more than three FMOs in FMO1–5 are indicated in bold text, including amino acid metabolism (Glycine, serine, and threonine metabolism; Cyanoamino acid metabolism; Aminoacyl-tRNA biosynthesis; Selenoamino acid metabolism; Taurine and hypotaurine metabolism; Cysteine and methionine metabolism; Arginine and proline metabolism; Lysine biosynthesis; Alanine, aspartate, and glutamate metabolism) and metabolism of cofactors and vitamins (Pantothenate and CoA biosynthesis; Vitamin B6 metabolism) (see also – ). (B) The levels of the top 25 changed metabolites between FMO1-OE cells and empty vector control cells are shown in heat map. (C) The levels of metabolites significantly regulated by FMO1 are shown in FMO1-OE cells and empty vector control cells.

Article Snippet: Mouse FMO1 (Accession No. U87456 ), FMO2 (Accession No. AF184981 ), and FMO3 (Accession No. NM_008030 ) were cloned from mouse liver cDNA (ZYAGEN) and inserted into Gateway TM pcDNA TM -DEST47 Vector (Invitrogen 12281010).

Techniques: Plasmid Preparation