Journal: bioRxiv

Article Title: Senescent cell death as an aging resistance mechanism in naked mole-rat

doi: 10.1101/2020.07.02.155903

Figure Lengend Snippet: a , RNA-seq expression level analysis of anti-apoptotic BCL2 family genes. Y-axis: fold changes of BCL2 family mRNAs in INK4a-transduced NMR-fibroblasts relative to mock-transduced NMR-fibroblasts from n = 3 biological replicates. b, c , Quantification of Annexin V-positive cells in INK4a-tranduced NMR-fibroblasts with human-BCL-XL ( n = 3 biological replicates) or NMR-BCL-XL ( n = 2 biological replicates) transduction (%). d , 20 days after INK4a transfection into fibroblasts, the cells were treated with Pan-caspase inhibitor, Z-VAD-FMK for 24 h and PI-positive cells were quantified (%). n = 4 biological replicates. e , 20 days after INK4a transfection into fibroblasts, the cells were treated with indicated doses of Fer-1 or DFO for 2h and PI-positive cells were quantified (%). f , qRT-PCR analysis for the expression of DDIT3 and GADD34 in INK4a-transduced NMR-fibroblasts. g , qRT-PCR analysis for the expression of DDIT3 and GADD34 in INK4a-transduced NMR-fibroblasts transduced shRNA for each gene. h , Quantification of Annexin V-positive cells transduced shRNA for each gene. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; t -test for a, d , and f ; one-way ANOVA followed by Tukey’s multiple comparison test for b and c ; one-way ANOVA followed by Dunnett’s multiple comparison test for e, g and h . Data are mean ± SD from n = 3 biological replicates for a, b, e, f, g and h .

Article Snippet: Each plasmid and packaging vectors (pCMV-VSV-G-RSV-Rev and pCAG-HIVgp) were used to transfect HEK293T cells with Polyethylenimine MAX transfection reagent (Polysciences), according to the manufacturer’s instructions.

Techniques: RNA Sequencing, Expressing, Transduction, Transfection, Quantitative RT-PCR, shRNA, Comparison

Journal: bioRxiv

Article Title: Senescent cell death as an aging resistance mechanism in naked mole-rat

doi: 10.1101/2020.07.02.155903

Figure Lengend Snippet: a , Quantification of PI-positive cells in NMR-fibroblasts treated with indicated doses of H 2 O 2 for 6 h. b , PCA plot of metabolome differences in mouse- or NMR-fibroblasts at 12 days after INK4a or mock vector transduction. c , Volcano plots of metabolome differences in mouse- or NMR-fibroblasts at 12 days after INK4a transduction compared to mock. d , Levels of serotonin (upper left), 5-hydroxyindole-3-acetic acid (5-HIAA) (lower left), kynurenine (upper right), and quinolinic acid (lower right) measured by LC-MS/MS in mouse- or NMR-fibroblasts at 12 days after INK4a transduction. Arrows indicate metabolic pathways of each metabolite. Serotonin metabolic pathway can generate H 2 O 2 . Data are mean ± SD from two technical replicates for each cell line ( n = 3 biological replicates). e , Levels of uric acid (upper) and allantoin (lower) measured by LC-MS/MS in mouse- or NMR-fibroblasts at 12 days after INK4a transduction. Arrows indicate metabolic pathways of each metabolite. These pathways can generate H 2 O 2 . XO; xanthine oxidase, XDH; xanthine dehydrogenase. Data are mean ± SD from two technical replicates for each cell line ( n = 3 biological replicates). f , 20 days after INK4a transfection into fibroblasts, the cells were treated with NAC for 24 h and PI-positive cells were quantified (%). Data are mean ± SD from n = 6 biological replicates. g , Schematic diagram representing SCD in NMR cells and cellular senescence in mouse cells. In NMR cells, inherent vulnerability to ROS and unique metabolic system induces RB-dependent SCD, which simultaneously results in multilamellar bodies accumulation and autophagy dysregulation. * P < 0.05; Two-way ANOVA followed by Sidak’s multiple comparisons test for a . t -test for d , e and f. Data are mean ± SD from n = 3 biological replicates except for d , e and f .

Article Snippet: Each plasmid and packaging vectors (pCMV-VSV-G-RSV-Rev and pCAG-HIVgp) were used to transfect HEK293T cells with Polyethylenimine MAX transfection reagent (Polysciences), according to the manufacturer’s instructions.

Techniques: Plasmid Preparation, Transduction, Liquid Chromatography with Mass Spectroscopy, Transfection