Journal: Cell Metabolism
Article Title: Obesity-Induced Cellular Senescence Drives Anxiety and Impairs Neurogenesis
doi: 10.1016/j.cmet.2018.12.008
Figure Lengend Snippet: ALISE Phenotype Drives CCF Accumulation and the SASP Similar to glia in the brains of obese mice, mouse adult fibroblasts (MAFs) show an accumulation of lipids in senescence (ALISE) phenotype. (A–F) Depletion of lipids from culture media reduces area of lipid droplets in MAFs. (A) The ALISE phenotype in MAFs is characterized by an increased area of lipid droplets surrounded by Plin2 vesicles (scale bars, 20 μm). (B) Quantification of Nile Red-positive staining in non-senescent young (you) and senescent (sen) MAFs (p < 0.0001, p = 0.0004). (C and D) Suppression of the ALISE phenotype reduces frequencies of cytoplasmic chromatin fragments (CCF) in senescent fibroblasts (p < 0.0001, p = 0.0006) (scale bars, 10 μm), but (E) and (F) does not affect number of 53BP1 DNA damage foci (p = 0.0973) (scale bars, 10 μm). (G–K) Senescent fibroblasts show increased secretion of SASP components including Il-6, KC (Cxcl1), and Ip-10 (Cxcl10) that is alleviated upon suppression of the ALISE phenotype. (G) Heatmap shows fold change in secretion of SASP components in cell culture media over a 72-hr time period when compared to non-senescent fibroblasts cultured with lipid-containing media. Each square represents a separate biological replicate, i.e., MAFs isolated from a different mouse donor. Concentration of SASP components in culture media with and without lipids (ALISE phenotype suppression) for (H) Il-6 (p = 0.0003, p < 0.0001), (I) Ip-10 (Cxcl10) (p = 0.0002, p = 0.0008), (J) Vegf (p = 0.6407, p = 0.003), and (K) Kc (Cxcl1) (p = 0.0114, p = 0.0075). (L) Images show accumulation of lipid droplets in senescent but not non-senescent GFAP-positive astrocytes (scale bars, 20 μm). (M–O) Characterization of senescence in astrocytes. Senescent astrocytes show (M) the ALISE phenotype measured using Nile Red staining (t (2.034) = 11.32, p = 0.0073), (N) increased frequencies of telomere dysfunction measured as the frequency of cells with 1 or more telomere associated DNA damage focus (TAF) (t (4) = 12.45, p = 0.0002), and (O) increased frequencies of CCF (t (2) = 7.551, p = 0.0171). Senescence was induced by X-ray irradiation (10Gy) and established within 14–21 days post-irradiation. Data are from n = 3–6 mice per group. Mean ± SEM plotted. ∗ p ≤ 0.05 and ∗∗ p ≤ 0.001.
Article Snippet: Anti-GFAP guinea pig polyclonal antibody (for IHF) , Synaptic Systems , Cat # 173 004; RRID: AB_10641162.
Techniques: Staining, Cell Culture, Isolation, Concentration Assay, Irradiation
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