Figure 3. Daphnia Sir2 exhibits functional activity similar to mammalian Sirt1. (A) Daphnia Sir2 overexpression confers cytoprotection to heat shock. Cos-1 cells were transfected with Daphnia Sir2 expression construct and heat shocked at 420 C for 20 and 30 minutes as indicated. Blue bars depict empty vector control and red bars represent the Daphnia Sir2 transfections. Percent cell death was measured using Trypan Blue viability assay 24 hours following heat shock. Error bars represent standard deviations. (B) Daphnia Sir2 enhances the transcriptional induction of genes regulated by a heat shock. Cos-1 cells were co-transfected either with the empty vector (blue bars) or Daphnia Sir2 expression construct (red bars), pGL4.41 (luciferase reporter plasmid, Promega), and pRL–Null (for normalization of transfection efficiency, Promega) and treated with CdCl2 at 24 hours following transfection. The luciferase assay was performed on cell extracts 6 hours following CdCl2 treatment. Error bars represent standard deviations. A Student T test of arcsin transformed proportions was used to determine statistical significance in A (* t stat: 3.98, df: 3, p: 0.007; ** t stat: 7.37, df: 3, p: 0.0003). In B an ANOVA was performed (F: 309, df: 2,6, p:9.2×10−5) followed by a post hoc Tukey test, both # and ## indicate differences between the means at p < 0.5.