1. Volume 46, Issue 2, Pages 187-199 (April 2012)
The Sam68 STAR RNA-Binding Protein Regulates mTOR Alternative Splicing during Adipogenesis 
Marc-Étienne Huot, Gillian Vogel, Amber Zabarauskas, Chau Tuan-Anh Ngo, Jasmin Coulombe-Huntington, Jacek Majewski, Stéphane Richard 
Molecular Cell 
Volume 46, Issue 2, Pages (April 2012)
DOI: /j.molcel
Copyright © 2012 Elsevier Inc. Terms and Conditions

2. Molecular Cell 2012 46, 187-199DOI: (10.1016/j.molcel.2012.02.007)
Copyright © 2012 Elsevier Inc. Terms and Conditions

3. Figure 1
Sam68−/− Mice Are Leaner Than Their Wild-Type Littermate Controls
(A) Sam68−/− or Sam68+/+ mice were maintained on a normal chow diet and weighed at 6, 12, and 24 weeks of age, as indicated. The bar denotes the mean and significance as determined by Student's t test. ∗∗p < and ∗p < 0.01.
(B) The fat pads were isolated from Sam68−/− or Sam68+/+ mice and weighed. EF, epididymal fat; IF, inguinal fat; RF, retroperitoneal fat; BF, brown fat; H, heart. Error bars represent ± SEM.
(C) Representative transverse MRI images are shown for mice 15–16 or 28 weeks of age. The percentage body WAT is indicated.
(D) The food intake for Sam68−/− or Sam68+/+ mice was calculated as daily average values adjusted to body weight. Error bars represent ± SEM.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 2 Sam68−/− Mice Are Protected from Dietary-Induced Obesity
(A and B) Sam68−/− or Sam68+/+ mice were fed either a chow diet (A) or a high-fat diet (HFD) (B) for 16 weeks. The weight of the mice is indicated and expressed as the mean body weight. Error bars represent ± SEM.
(C) Mice fed a HFD were starved overnight and then given orally a glucose bolus (dose), and blood glucose was then monitored with time. Error bars represent ± SEM.
(D) Insulin tolerance tests (ITTs) were performed on anesthetised males by intraperitoneal administration of insulin, and blood glucose was monitored with time. Error bars represent ± SEM.
(E) Mice fed a HFD were starved overnight and then challenged with insulin for 5 min. Activation of the insulin receptor (IR) was assessed by immunoblotting liver extracts with anti-pY1162/3 IR antibodies. Total IR serves as a loading control.
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5. Figure 3 Sam68 Is Required for Adipogenesis of 3T3-L1 Cells
(A) 3T3-L1 cells were transfected with siGFP (control) or siSam68. 3T3-L1 cells or siSam68-treated 3T3-L1 cells were transfected with pcDNA3.1 (control) or an expression vector encoding hSam68. Adipocyte differentiation was assessed by oil red O staining.
(B and C) 3T3-L1 cells were stably transfected with pRetrosuper or Sam68sh pRetrosuper. A bulk population of pRetrosuper and two individual clones (Sam68sh2 and Sam68sh22) were selected for analysis by immunoblotting using Sam68 and actin antibodies, and adipogenesis was analyzed as described in (A).
(D) The mRNA levels of PPARγ, aP2, and Glut4 normalized to GAPDH were assessed by RT-qPCR. The data are expressed as relative values from day 0 and day 8 differentiation of pRetrosuper and Sam68sh2. Error bars represent ± SEM.
(E) Relative number of ADSCs isolated from WAT tissue of 6- to 7-month-old wild-type and Sam68−/− mice (n = 3). Error bars represent ± SEM; ∗p <
(F) Expression levels of pericyte markers Vcam1 and PDGFRβ by RT-qPCR in ADSCs isolated from wild-type and knockout WAT (n = 2). Fold induction is normalized to 18S rRNA levels. Sam68 serves as a control. Error bars represent ± SEM.
(G) Expression levels of pericyte markers NG2 and α-SMA by RT-qPCR in total fat deposits isolated from wild-type and knockout mice (n = 3). Fold induction is normalized to 18S rRNA levels. Sam68 serves as a control. Error bars represent ± SEM.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 4
Sam68 Inactivation Increases the Retention of Intron 5 of mTOR mRNA during Adipogenesis
(A) Splicing of mTOR exon 4–6 schematic and the intron 5 retention leading to the generation of mTORi5.
(B) The mRNA levels of mTORi5, mTORexon45-46, PPARγ, and Sam68 normalized to PRMT1 as assessed by RT-qPCR from control pRetrosuper (pRetrosuper) and Sam68 knockdown (Sam68sh) 3T3-L1cells at 0 and 4 days of differentiation. Error bars represent ± SEM.
(C) Schematic of the mTOR minigene and its validation. Detection, by RT-PCR, of mTORexon4-6, mTORi5, and mTORexon4 in wild-type MEFs after knockdown of Sam68 (siSam68) or overexpression of Sam68 (myc-Sam68). mTORexon4 and GAPDH serve as internal expression controls.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 5 Sam68 Associates with RNA Sequences within mTOR Intron 5
(A) Schematic of the mTOR minigene in its original form followed by the different mutations in the Sam68-binding sequences previously identified.
(B) Sequence of intron 5 SBS#1 and SBS#2, as well as the mutated (UUUCA) versions. Peptide pull-down assays were performed using 3T3-L1 cells and immunoblotted with Sam68 antibodies.
(C) ChIP analysis using anti-Sam68 or control IgGs. Bound DNA was analyzed in triplicate by qPCR with the indicated primers specific for the mTOR gene. Mean values are expressed as fold enrichment. Error bars represent ± SEM.
(D) Minigene assays show the requirement for SBS#1 and SBS#2. Fold inclusion of intron 5 is expressed as the level of mTORi5 over mTORexon4 and normalized with the total level of the minigene expression product (mTORexon4). Error bars represent ± SEM.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 6
Sam68-Deficient Cells Exhibit Decreased mTORC1 and mTORC2 Activity
(A) Cellular extracts were immunoblotted with the indicated antibodies during adipocyte differentiation. Tubulin and S6 served as loading controls, and the molecular markers are shown on the left in kilodaltons (kDa). The relative quantification is shown below the panels.
(B) pRetrosuper or Sam68sh2 3T3-L1 cells display impaired mTORC1 and mTORC2 activity following insulin stimulation. Starved cells (16 hr) were left untreated or treated with rapamycin and/or insulin 15 min prior to stimulation. Cell extracts were prepared and immunoblotted with the indicated antibodies.
(C) Sam68sh 3T3-L1 cells were transiently transfected with empty vector or an mTOR expression vector and induced to differentiate for 6 days. Transfections were repeated 1 day after differentiation initiation and adipogenesis visualized by oil red O staining.
(D) Quantification of percentage of cells positive for oil red O staining. The graph is representative of >10 fields. Error bars represent ± SEM.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2012 Elsevier Inc. Terms and Conditions