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. Author manuscript; available in PMC: 2009 Sep 14.
Published in final edited form as: Cancer Cell. 2008 May;13(5):454–463. doi: 10.1016/j.ccr.2008.03.004

Figure 6. SirT1-Related Effects of Tenovins in Mammalian Cells.

Figure 6

(A) MCF7 cells were transfected with the RGC-ΔFos-LacZ p53-dependent reporter construct as well as a control vector or an expression vector for the SirT1-363Y dominant negative mutant. All samples were also transfected with a control plasmid expressing luciferase under the control of the SV promoter. β-galactosidase activity was measured 32 hr after transfection and values were normalized using the luciferase readings. Values correspond to three independent experiments ± SD.

(B) H1299 cells (p53 null) were transfected with vectors expressing p53 and mdm2 in the absence or presence of a vector expressing SirT1 (pCMV-SirT1). Cells were treated with increasing concentrations of tenovin-1 for 6 hr, and the levels of p53 and SirT1 were analyzed by western blot using DO1 and antibody 2G1-F7 (Cat. No. 05-707, Upstate), respectively. Note that pCMV-SirT1 encodes SirT1 isoform-1. Endogenous SirT1 isoform-1 was also detected in lanes 1 through 5 upon longer exposure of the blots. The band below ectopic SirT1 could correspond to a SirT1 isoform.

(C) MCF-7 cells were treated with 10 μM tenovin-1 for the indicated times and analyzed by western blotting using an antibody against K382-acetylated p53 (Cat. No. 614202, BioLegend) or the DO1 antibody against the N terminus of p53. PCNA was detected as a loading control.

(D) H1299 cells transfected with a vector for p53 were treated for 6 hr with the indicated concentrations of tenovin-6. K382-acetylated p53 and total p53 were detected as above.

(E) H1299 cells were transfected with a vector for p53 expression (upper panels) or p53R273H (lower panels) in the absence or presence of pCMV-SirT1. Cells were treated for 6 hr with the indicated concentrations of tenovin-1. K382-acetylated p53 and total p53 were detected.

(F) H1299 cells were transfected with a vector for wild-type p53 expression (lanes 1, 2, and 3) or p53R273H (lanes 4, 5, and 6). Cells were left untreated (lanes 3 and 4) or treated with 10 μM (lanes 2 and 5) or 20 μM (lanes 1 and 6) tenovin-6 for 6 hr. K382-acetylated p53 and total p53 were detected, and the ratio between the amount of K382-acetylated p53 and the total amount of p53 in each lane was calculated. Note that these ratios are relative and do not correspond to the actual fraction of acetylated p53 in cells. Lanes 7, 8, and 9 correspond to loading 1/10 of the amount of protein in samples in lanes 4, 5, and 6, respectively.

(G) H1299 cells were transfected with a vector for wild-type p53 expression (lanes 1 through 3) or p53R273H (lanes 4 through 6) in the absence (lanes 1 and 4) or presence (lanes 2, 3, 5, and 6) of ectopic mdm2. In lanes 3 and 6, cells were treated for 6 hr with 10 μM tenovin-1. Total p53 was detected with DO1 antibody. β-gal expression was used as a transfection efficiency and loading control.

(H) H1299 cells were treated with 10 μM tenovin-1 for the indicated times. Endogenous p14ARF was detected using a mouse monoclonal antibody (Ab-3 14P03, Neomarkers). PCNA was detected as a loading control.