Computational protocol: Targeting of NAT10 enhances healthspan in a mouse model of human accelerated aging syndrome

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Protocol publication

[…] Isolation and culture of adult mouse fibroblasts from skin and lungs were performed using an established protocol. Cells were washed with PBS and fixed for 10 min with 4% PFA in PBS. Cells were permeabilised for 5 min with PBS/0.2% Triton X-100 and blocked with PBS/0.2% Tween 20 (PBS-T) containing 5% BSA. Coverslips were incubated for 1 h with primary antibodies and for 30 min with appropriate secondary antibodies coupled to Alexa Fluor 488 or 594 fluorophores (Life Technologies), before being incubated with 2 μg/ml DAPI. Pictures were acquired with a FluoView 1000 confocal microscope (Olympus) and images were quantified using ImageJ. All the immunofluorescence experiments were performed independently at least three times and the pictures shown in the figures are representative images of at least three experiments. [...] All major organs were isolated following killing and then fixed in 10% formalin overnight. On the second day the fixed organs were transferred to 70% ethanol, were placed in cassettes, embedded in paraffin and serial 5 μm sections were collected on Superfrost Plus slides (Fisher) using a Leica microdissection system (LMD7000). Hematoxilin and eosin (HE) and immunohistochemistry staining were performed using standard protocols. The organs were examined for abnormalities by a Board Certified Veterinary Pathologist (MJ). Sections of heart containing aortic outflow, pulmonary artery and myocardium were stained using a primary antibody against alpha smooth muscle actin (SMA-Sigma Aldrich Cat No: A2547 1:1000) and a secondary antibody (Alexa 488 Life Technologies at 1:100). The heart sections were also stained using DAPI alone and with an antibody against Acetyl α Tubulin (K40) (5335 Cell Signaling). Representative histology images were obtained from whole slide images scanned on a Hamamatsu NanoZoomer in brightfield and fluorescence modes. The thickness of the subcutaneous fat layer in the skin and nuclei number in the aorta were measured using whole slide images and the Hamamatsu NDP view software at a magnification of ×200 (resolution of 0.45 μm per pixel). The skin (always the same region/the flanks) and aorta regions for analysis were identified by a pathologist (MJ) and manually annotated using the HALO image analysis software (Indica Labs). The cells inside these annotated regions were identified and counted using the HALO software CytoNuclear v1.5 algorithm, the output of cell density (cells/mm2) was used to differentiate between the treatment groups. SMA intensity quantification was performed using ImageJ. [...] RNA was extracted as described above and quality control assessed using the 2100 Bioanalyzer (Agilent Technologies). Because of financial constrains we used n = 2 mice/group. Transcriptome data was obtained using paired end sequencing, with read lengths of 150 bp, on a NextSeq 500 machine. Trimmed reads were aligned using STAR aligner (version 2.4.2a) to the mouse genome assembly GRCm38. Normalization of the read counts and differential expression analysis was performed using three commonly used software programs: DeSeq2, edgeR, and Cuffdiff 2. Our conservative approach defined genes differentially expressed as those found to be in common between the results of at least two of the three software programs mentioned above. The log-2 fold change in gene expression presented in Fig.  and Fig.  correspond to the log-2 fold change returned by DeSeq2. DeSeq2 and edgeR used as an input raw read counts produced by featureCounts from the Bioconductor (version 3.3) Rsubread package (14) in R version 3.3.1. For the DeSeq2 and edgeR analyzes, we filtered out genes that had 0 or 1 read support across all samples. In the DeSeq2 differential expression analysis, we selected genes that were up or down regulated at a FDR lower than 0.1. In edgeR differential expression analysis, we selected genes up or down regulated with a p-value less than 0.05. Gene ontology analysis was performed using the mouse genome informatics visual annotation display. For the analysis of the biological term fold enrichment, a ratio between the frequency of genes in our set to frequency of genes in the whole genome was calculated. […]

Pipeline specifications

Software tools ImageJ, HALO
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Mus musculus, Homo sapiens
Diseases Cardiovascular Diseases, Progeria, Genetic Diseases, Inborn