(a) Luciferase assay with Sirt1-7 promoter sequences in pGL3 vector
shows increased Sirt3, 4 & 5 luciferase activity in G4/p53 −/−
compared to G4 /p53 +/+ MEFs (pGL3 vector and p21 promoter serve as background
and positive controls); (b) Luciferase assays with Sirt1-7 3’UTR reveals
increased luciferase activity in G4/p53 −/− MEFs compared to
G4/p53 +/+ MEFs; (c) Polysome analyses in WT, p53 −/−, G4/p53 +/+,
and G4/p53 −/− MEFs shows increased polysome occupancy of Sirt1,
2, 6, 7 transcripts in G4/p53 −/− MEFs (two independent
experiments); (d) Western blot analysis of MEFs treated with proteasome
inhibitor MG132 indicates that Sirt7 protein abundance is also regulated by
proteasome-mediated degradation; (e) RT-qPCR analysis of WT MEFs transduced with
PGC-1α - expressing adenovirus shows that PGC-1α overexpression in
MEFs induces Sirt3, 4 & 5 mRNA levels; (f) Western blot analysis of MEFs
overexpressing PGC-1α or GFP demonstrates that PGC-1α increases
Sirt3, 4 & 5 protein abundance without affecting other sirtuins. Results are
expressed as mean ± s.e.m. and are derived from three independent
experiments in two MEF cell lines/genotype unless stated otherwise; t-test was
used to determine statistical significance with p <0.05 considered as
significant, as indicated by (*).