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. 2011 Dec 21;109(4):E187–E196. doi: 10.1073/pnas.1105304109

Fig. 1.

Fig. 1.

c-MYC induces SIRT1 protein expression. Lysates were prepared at the indicated time points and subjected to Western blot analysis (A and D–G). Detection of α-tubulin or β-actin served as loading controls. (A) RAT1 c-myc−/− (HO15) fibroblasts (82) stably expressing c-Myc-ER were starved for 48 h at 0.1% FBS and then treated with 300 nM 4-hydroxytamoxifen (4-OHT) for activation of Myc-ER. As a control, serum starvation was continued for another 48 h (right lane). (B) RAT1 c-myc−/− (HO15) fibroblasts stably expressing c-Myc-ER were starved for 48 h at 0.1% FBS and then treated with 300 nM 4-OHT for activation of c-Myc-ER. At the indicated time points mRNA expression of c-MYC target genes and SIRT1 was analyzed by quantitative PCR (qPCR) [SIRT1 primer pair 1 (p1) and 2 (p2)]. Western blot analysis of these cells is shown in A. Bars represent mean values of biological triplicates (SIRT1) or duplicates with SE. LDHa, lactate dehydrogenase A; NPM, nucleophosmin; ODC, ornithine decarboxylase. (C) qPCR analysis of SIRT1 expression and direct c-MYC target genes 11 h after c-MYC activation in P493-6 cells, a pre–B-cell line that harbors a conditional c-MYC allele (83). Bars represent mean values of biological triplicates with SE. CDK4, cyclin-dependent kinase 4; DKC, dyskerin. Five additional cell lines were analyzed with similar results after induction of a conditional c-MYC allele or after activation of endogenous c-MYC with up to two different SIRT1-specific primer pairs (SI Appendix, Fig. S1 C and D). (D) LS-174T colorectal cancer cells were treated with 1 μg/mL DOX for the indicated periods to activate the expression of c-MYC–specific shRNA (84). (E) c-myc+/+ (TGR) and c-myc−/− RAT1 fibroblasts (HO15) were kept for 48 h at 0.1% FBS and then restimulated with 8% (vol/vol) FBS for the indicated periods. (F) HDF expressing either SIRT1-GFP or GFP were serum starved at 0.1% FBS for 48 h and then restimulated with 10% (vol/vol) FBS for the indicated periods. See also SI Appendix, Fig. S1E. (G) HO15 (myc−/−) and TGR-1 (myc+/+) cells were treated with MG132 (10 μM) for the indicated periods. (H) Expression of c-MYC and SIRT1 protein in colorectal cancer and adjacent normal colonic cells. Shown are representative results obtained by immunohistochemical analysis of consecutive sections derived from one colorectal biopsy (of 15) with antibodies directed against c-MYC and SIRT1. (Magnification: 200×.) N, normal tissue; T, tumor. See also SI Appendix, Fig. S2.