Fig. 3

SIRT6 knockout neurons resist apoptosis and the effects of nicotine. a Schematic representation of the SIRT6 conditional strain of mice. Exons 2 and 3 (which comprise the enzymatic active site) of SIRT6 are flanked with lox-P sites, and when cre-recombinase is expressed, these exons are excised. b Schematic representation of SIRT6 conditional overexpressing (OX) transgenic allele. The chicken β-actin promoter is followed by a lox-P flanked stop site in all six reading frames, followed by SIRT6 gene cDNA and poly-A signal. For both KO and OX schemes, cre-recombinase is expressed from the nestin promoter, which allows creation of brain-specific SIRT6 knockout (BSKO) or brain-specific overexpression (BSOX) animals. c Typical western blot analysis of cortex lysates from BSKO or BSOX mice and their WT littermates. d Bar graphs representing surviving fraction of primary WT, KO, and OX neurons, cultured in vitro, 24 h after their stress with various insults. Neurons lacking SIRT6 resist apoptosis after stress. Con – control, MPP⁺ − 1-methyl-4-phenylpyridinium (500 μM), SS – starvation (in case of primary neurons, B27 serum and bFGF were withheld), MG – proteasome inhibitor MG132 (10 μM). Survival of cells was measured using flow cytometry, after cells had been stained with the apoptotic markers– Annexin-V and propidium iodide (PI). A cell was considered “alive” if it was negative for both Annexin-V and PI staining. Mean ± SEM, n = 3 independent experiments with at least 10,000 cells analyzed in each experiment for each treatment; two-tailed t-test analysis *p < 0.05, **p < 0.01, ***p < 0.001. e Survival of KO and OX neurons, respectively, pre-treated with 1 μM nicotine for two hours before MPP⁺ stress. WT cells pre-treated with nicotine had improved survival under stress, while SIRT6 KO and OX cells did not benefit further from nicotine pretreatment. f Representative flow cytometry plots showing WT, SIRT6 KO, and OX neurons starved and stained with Annexin-V and PI, included in analyses depicted in D. Each dot represents a single cell. Dot coloring reflects local cell density in the given area of the graph