(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 Dicer^flox/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 Dicer^Flox/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.

(A) Luciferase assays were performed 24 h after transfection of the full-length 1.6 kb SIRT1 3'-UTR downstream of luciferase (SIRT1 3'-UTR) or constructs with 4 bp in the seed-binding regions mutated (SIRT1 3'-UTR 181mt, left panel; SIRT1 3'-UTR 9mt, right panel) and control, miR-181a, b, and c miRNA mimics (left panel) or pSuper and pSuper miR-9 expression constructs (right panel). Data are mean ± s.d. for eight experiments. (B-C) mESCs were transfected with individual miRNA expression constructs; protein and RNA were isolated 48 h later. (B) Repression of SIRT1 protein was analyzed by western blotting. Data are representative of six experiments. (C) qRT-PCR analysis of SIRT1 mRNA levels and mature miRNA levels. Data are mean ± s.d. for four samples.

(A) mESCs, MEFs, and iPS cells were subject to western blot analysis with antibodies to the indicated proteins. (B) SIRT1, HDAC1, and HDAC2 protein levels relative to tubulin were quantified by densitometry. (C) qRT-PCR analysis of SIRT1 mRNA levels in mESCs, MEFs, and iPS cells were measured relative to GAPDH. (D) qRT-PCR analysis of miRNA expression relative to miR-16 in mESCs, MEFs, and iPS. Data are mean ± s.d. for three samples.

(A-B) Protein and RNA were extracted from mESC and tissues from ~6-week-old mice. (A) Western blot analysis with antibodies against SIRT1 (Frye antiserum top blot; Upstate antiserum lower blot), HDAC1, HDAC2, and tubulin. (B) qRT-PCR analysis of SIRT1, HDAC1, and HDAC2 normalized to GAPDH levels. Data are mean ± s.d. for four samples. (C-D) Protein and RNA were isolated from mESCs differentiated in vitro for up to 20 days (EBs d2-20). (C) Western blots analysis of expression of SIRT1, various HDACs, markers of pluripotent embryonic stem cells, and markers of differentiation. Data are representative of four experiments. (D) qRT-PCR analysis of SIRT1, HDAC2, markers of pluripotent embryonic stem cells, and markers of differentiation. Data were normalized to GAPDH and plotted as expression relative to the mean of four mESC samples. Data are mean ± s.d. for four samples.

(A) 18 miRNAs from nine miRNA families that potentially target the 3'-UTR of SIRT1 were induced during mESC differentiation at the time SIRT1 protein was downregulated. Their fold induction in d20 embryoid bodies above their expression in undifferentiated mESCs was plotted on the y-axis, and the location of their seed binding site in the 3'-UTR of mSIRT1 was plotted on the x-axis. (B-C), qRT-PCR of miRNA expression relative to miR-16 from undifferentiated mESCs and differentiating embryoid bodies of specific miRNAs that potentially target SIRT1. Data are mean ± s.d. for four samples.

(A-C) mESCs were differentiated and transfected at d4 and d7 with LNA probes. Protein and RNA were isolated on indicated days. (A) qRT-PCR of miR-9 shows the expected upregulation during differentiation and 35% inhibition when embryoid bodies were transfected with LNA-miR-9 but not with LNA-SCR. (B) qRT-PCR show no significant change in SIRT1 mRNA levels. Data are mean ± s.d. for four samples and representative of three experiments. (C) Western blot analysis shows that the downregulation of SIRT1 protein during mESC differentiation was specifically inhibited in cells transfected with LNA-miR-9 but not by transfection of LNA-SCR or untransfected controls. Data are representative of four experiments. (D-F) EBs were dissociated and transfected at d6 with LNA probes. Protein and RNA were isolated on d11. (D) Western blot analysis shows upregulation of SIRT1 protein in EBs transfected with LNA-miR-9 but not LNA-SCR. (E) qRT-PCR analysis shows inhibition of miR-9 in EBs transfected with LNA-miR-9, but not with LNA-SCR, and no significant change in SIRT1 mRNA levels (F). Data are mean ± s.d. for four samples.