Research Paper Volume 2, Issue 7 pp 415—431

miRNAs regulate SIRT1 expression during mouse embryonic stem cell differentiation and in adult mouse tissues

Figure 2. miRNAs post-transcriptionally regulate SIRT1.. (A) mESCs were differentiated and treated on d8 with the proteasome inhibitor MG-132 (10 μM, 3-7 h), and protein lysates were analyzed on western blots. Data are representative of four experiments. (B) Protein levels of SIRT1 and REST relative to tubulin levels were quantified by densitometry with NIH Image. (C-E) The consequences of Dicer inactivation and loss of small RNAs were assessed in protein lysates and RNA from livers of control and Dicerflox/flox mice injected with the AAV8 vector expressing cre at the indicated times. (C) Western blotting was used to analyze 70 μg of liver lysate and 10 μg of mESC lysate. (D) SIRT1 protein levels relative to tubulin or GAPDH were quantified by densitometry. (E) SIRT1 and Dicer mRNA levels were measured by qRT-PCR. Data are mean ± s.d. for four samples. (F-H) Lung fibroblasts were cultured from DicerFlox/Flox mice and infected with adenoviral Cre or GFP. (F) SIRT1 protein levels were measured by western blotting 72 h after Cre inactivation of Dicer. (G) SIRT1 protein levels relative to tubulin were quantified by densitometry. (H) mRNA levels of SIRT1 and Dicer were measured by qRT-PCR. Data are mean ± s.d. for three samples. (I-K) siRNAs were transfected into NIH3T3 cells to knockdown DGCR8, Dicer, or GL3 luciferase as a control. (I) DGCR8 knockdown and increased SIRT1 protein levels were analyzed by western blotting 72 h after siRNA transfection. Data are representative of three experiments. (G) qRT-PCR analysis confirmed Dicer knockdown and no significant change in SIRT1 mRNA levels. Data are mean ± s.d. for three samples.