Research Paper Volume 12, Issue 19 pp 18942—18956

(A) 3T3-L1 cell lines created for this study. Using lentiviral mediated gene expression and shRNA knock-down techniques, 3T3L1 cells were infected with shNegative (non-targeted shRNA expressing) lentivirus, p53DN (dominant-negative p53 expressing) lentivirus, shSirt1 (Sirt1 targeted shRNA expressing) lentivirus, and shSirt1+LKB1-K48R (LKB1 K48R mutation) lentivirus. Infected cells were selected by corresponding antibiotics (blasticidin or hygromycin). After confirmation of lentiviral expression, these preadipocyte cell lines were induced to become adipocytes as shown in the following figures. (B) SA-β-Gal staining time course shNegative vs. shSirt1. Sirt1 knock-down significantly increased SA-β-Gal expression at PID 15 (15 days after starting differentiation). (*p<0.05, n=12). (C) SA-β-Gal staining at PID 15. Dominant-negative p53 expression decreased basal 3T3-L1 SA-β-Gal expression (*p<0.05 vs. shNegative, n=12). Expression of the Sirt1 insensitive LKB1 K48R-mutant prevented Sirt1 knock-down effects. (Not Significant to shNegative). Representative figure is shown.