Fig. 4
Nicotine reverses stress induced SIRT6 activity and inflammation. a Surviving fraction of WT and SIRT6 KO fibroblasts, cultured in vitro, 24 h after their stress with various insults. Fibroblasts lacking SIRT6 resist apoptosis after stress. Con – control, MPP+ − 1-methyl-4-phenylpyridinium (500 μM), SS – starvation (fetal bovine serum was withheld), MG – proteasome inhibitor MG132 (10 μM), Roten – rotenone (10 μM), Etop – etoposide (20 μM). Survival of fibroblasts 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. b Typical western blot analysis of WT and SIRT6 KO fibroblasts. KO fibroblasts have higher levels of pAKT (S473) at baseline. c Survival of fibroblasts pre-treated with 1 μM nicotine for two hours before etoposide stress. WT cells pre-treated with nicotine had improved survival under stress, while SIRT6 KO cells did not benefit further from nicotine pretreatment. d Representative flow cytometry plots showing WT and SIRT6 KO fibroblasts stained with Annexin-V and PI, included in analyses depicted in C. Each dot represents a single cell. Dot coloring reflects local cell density in the given area of the graph. Survival of WT cells but not SIRT6 KO cells is improved by nicotine. e Typical western blot analysis of WT fibroblasts stressed with serum starvation. f Western blot analysis of WT fibroblasts stressed with MG132 (10 μM). SIRT6 increases under both SS and MG132 stress with a concomitant decrease in pAKT. g Typical western blot analysis of WT neurons treated with nicotine and or MPP+, as depicted in Fig.3e. Note the increase in SIRT6 from MPP+ stress and the lower levels under nicotine treatment. h Bar graph quantification of SIRT6 levels as depicted in G. i Typical western blot of WT neurons starved (of B27 and FGF) and treated with nicotine for 1.5 h (0.1, 1, 10, 100, and 1000 μM). SIRT6 increases after starvation but decreases upon nicotine exposure. j Bar graphs showing secretion of TNFα by primary neurons, measured by ELISA, 24 h after incubation with 1 μM nicotine. SIRT6 KO neurons secrete less TNFα than WT and are unaffected by nicotine. Mean ± SEM, n = 4 independent experiments, two-tailed T-test analysis for *, two-way ANOVA: pgenotype = 8.5•10− 6, pnicotine = 8.8•10− 3, pgenotype x nicotine = 1.6•10− 2