Journal: Cell Reports

Article Title: A Wars2 Mutant Mouse Model Displays OXPHOS Deficiencies and Activation of Tissue-Specific Stress Response Pathways

doi: 10.1016/j.celrep.2018.11.080

Figure Lengend Snippet: Upregulation of Mitochondrial Biogenesis Prevents Mitochondrial Respiratory Chain Dysfunction in Wars2 V117L/V117L MEFs, Skeletal Muscle, and iWAT (A and B) Oxygen consumption rate (OCR) (A) and extracellular acidification rate (ECAR) (B) were measured in cultured primary mouse embryonic fibroblasts harvested from Wars2 V117L/V117L and Wars2 +/+ embryos using a Seahorse XF24 analyzer. OCR and ECAR measurements were taken at baseline and following oligomycin (Oligo), carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), and rotenone and antimycin (Rot/Anti) treatment. OCR and ECAR measurements were normalized to live cell number. (C) Relative oxygen consumption rates of basal respiration, proton leak, ATP production, maximal respiration, and spare respiratory capacity in Wars2 +/+ and Wars2 V117L/V117L MEF cultures. There were 9 replicates of each genotype; mean ± SD. Data were log transformed and analyzed using an unpaired two-tailed t test or a Mann-Whitney t test. (D) MEFs were stained with MitoTracker green, and fluorescence in 30,000 cells per sample was quantified by fluorescence-activated cell sorting (FACS). There were 3 replicates of each genotype; mean fluorescence ± SD. Data were analyzed using an unpaired t test. (E) Relative mRNA expression analysis of Pgc1α in Wars2 +/+ and Wars2 V117L/V117L MEF cultures. There were 3 replicates of each genotype; mean fluorescence ± SD. Data were analyzed using an unpaired t test. (F–H) Relative mRNA expression analysis of (F) Pgc1α , (G) Tfam , and (H) Pparα in tissues harvested from Wars2 V117L/V117L and Wars2 +/+ male mice at 12 months of age. Wars2 V117L/V117L and Wars2 +/+ animal numbers were 5 and 5, respectively; mean ± SD. Data are shown as log transformed and analyzed using an unpaired two-tailed t test or a Mann-Whitney t test (skeletal muscle and iWAT for Pgc1α ). ∗ p

Article Snippet: Taqman probes used in this study: Wars2 (Exon 2-3) ( Mm04208965_m1), Wars2 (Exon 4-5) ( Mm04208967_m1), Wars2 (Exon 5-6) ( Mm00840490_m1), Pgc1α ( Mm01208835_m1), Atf4 ( Mm00515324_m1), Atf5 ( Mm00459515_m1), Chop ( Mm01135937_g1), Fgf21 ( Mm00840165_g1), Tfam ( Mm00447485_m1), Pparα ( Mm00440939_m1), Ucp1 (Mm01244861_m1), Dio2 ( Mm00515664_m1), Cidea ( Mm00432554_m1), Pparγ ( Mm00440945_m1), Cox7a1 ( Mm00438297_g1) and Cox8b ( Mm00432648_m1).

Techniques: Cell Culture, Transformation Assay, Two Tailed Test, MANN-WHITNEY, Staining, Fluorescence, FACS, Expressing, Mouse Assay

Journal: Biomedicines

Article Title: IGF1R Deficiency Modulates Brain Signaling Pathways and Disturbs Mitochondria and Redox Homeostasis

doi: 10.3390/biomedicines9020158

Figure Lengend Snippet: Effects of IGF1R deficiency on mitochondrial biogenesis and protein levels of brain mitochondrial genes. ( a ) Western blot analyses of nuclear factor E2-related factor 1 (NRF1), ( b ) mitochondrial transcription factor A (TFAM), ( c ) NADH dehydrogenase subunit 1 (ND1) and ( d ) mitochondrial-encoded cytochrome c oxidase 1 (MTCO1) protein levels. Bars represent the mean ± SEM of the quantification of samples from 6–9 mice per genotype after normalization with β-actin levels and respect to Igf1r fl/fl control mice. Representative immunoblots for each protein and their respective β-actin results are shown below the graphs. Dotted lines on Western blot images symbolize some removed interspacing lanes for a side-by-side display of samples from both groups. A * p

Article Snippet: Antibodies were obtained from the following sources: Akt (1:1000, BD Biosciences, San Jose, CA, USA (610861)); pAkt (Ser473) (1:1000, Cell Signaling, Danvers, MA, USA (4051)); CREB (1:750, Cell Signaling, Danvers, MA, USA (9197)); pCREB (Ser133) (1:1000, Millipore, Darmstadt, Germany (06-519)); DRP1 (1:1000, BD Biosciences, San Jose, CA, USA (611113)); pDRP1 (Ser616) (1:1000, Cell Signaling, Danvers, MA, USA (3455)); MAPK (ERK1/2) (1:1000, Cell Signaling, Danvers, MA, USA (9102)); pMAPK (ERK1/2) (Thr202/Tyr204) (1:1000, Cell Signaling, Danvers, MA, USA (4377)); pGSK3β (Ser9) (1:1000, Cell Signaling, Danvers, MA, USA (9336)); pGSK3β (Y216) (1:1000, Santa Cruz Biotechnology, Heidelberg, Germany (sc-135653)); GSK3β (1:1000, Santa Cruz Biotechnology, Heidelberg, Germany (sc-81462)); IGF-1R β(H60) (1:1000, Santa Cruz Biotechnology (sc-9038)); IR β(4B8) (1:1000, Cell Signaling, Danvers, MA, USA (3025)); ND1 (C-18) (1:1000, Santa Cruz Biotechnology, Heidelberg, Germany (sc-20493)); NRF2 (1:1000, Abcam, Cambridge, UK (ab31163)); NRF1 (1:1000, Santa Cruz Biotechnology, Heidelberg, Germany (sc-33771)); TFAM (1:750, Santa Cruz Biotechnology, Heidelberg, Germany (sc-23588)); Mfn1 (1:1000, Santa Cruz Biotechnology, Heidelberg, Germany (sc-50330)); Mfn2 (1:1000, Santa Cruz Biotechnology, Heidelberg, Germany (sc-100560)); MTCO1 (1:1000, Abcam, Cambridge, UK (ab14705)); OPA1 (1:1000, BD Biosciences, San Jose, CA, USA (612607)); PI3K (p110-alpha-C73F8) (1:1000, Cell Signaling, Danvers, MA, USA (4249S)); PSD95 (1:1000, Cell Signaling, Danvers, MA, USA (D27E11)); synaptophysin (1:1000, Sigma-Aldrich, St. Louis, MO, USA (S5768)); SNAP25 (1:1000, Sigma-Aldrich, St. Louis, MO, USA (S5187)); pTau (Ser 396) (1:1000, Santa Cruz Biotechnology, Heidelberg, Germany (sc-101815)); Tau (1:750, Sigma-Aldrich, St. Louis, MO, USA (T9450); actin (1:5000, Sigma-Aldrich, St. Louis, MO, USA (A5441)).

Techniques: Western Blot, Mouse Assay