1. Volume 18, Issue 1, Pages 51-61 (July 2013)
An SREBP-Responsive microRNA Operon Contributes to a Regulatory Loop for Intracellular Lipid Homeostasis 
Tae-Il Jeon, Ryan M. Esquejo, Manuel Roqueta-Rivera, Peter E. Phelan, Young-Ah Moon, Subramaniam S. Govindarajan, Christine C. Esau, Timothy F. Osborne 
Cell Metabolism 
Volume 18, Issue 1, Pages (July 2013)
DOI: /j.cmet
Copyright © 2013 Elsevier Inc. Terms and Conditions

2. Cell Metabolism 2013 18, 51-61DOI: (10.1016/j.cmet.2013.06.010)
Copyright © 2013 Elsevier Inc. Terms and Conditions

3. Figure 1 The miR-96/182/183 Locus Is Directly Regulated by SREBP-2
(A) qPCR analysis of miR-96, miR-182, and miR-183 in RNA from mice fed chow (N), chow supplemented with cholesterol (Ch), or chow supplemented with lovastatin plus ezetimibe (LE). Samples were normalized to sno202 RNA samples analyzed in parallel. Data are plotted relative to normalized values from the chow group set at 1.0.
(B) ChIP analysis for SREBP-2 binding in hepatic chromatin from Ch- versus LE-supplemented mice. The three regions in the miR-96/182/183 locus that were analyzed for SREBP-2 association are shown by the location of forward (F) and reverse (R) primer pairs 1, 2, or 3 used for the qPCR analysis as indicated. The thick gray box denotes the putative promoter region for the locus interrogated by primer pair 1, as discussed in the Results.
Data are represented as mean ± SEM. See also Figure S2.
Cell Metabolism  , 51-61DOI: ( /j.cmet )
Copyright © 2013 Elsevier Inc. Terms and Conditions

4. Figure 2 SREBP-2 Activates the miR-96/182/183 Promoter
The putative promoter region shown in gray at the bottom of Figure 1 was cloned upstream of luciferase in the control luciferase reporter, as shown and described in the Experimental Procedures. Key putative transcription-factor-binding sites that are conserved between mice and humans (Figure S3) are noted on the diagram of the sequence. Top, there are two E box motifs that are putative SREBP response elements, and point mutations were engineered into each separately or in combination, as noted by the X. Bottom, wild-type and the indicated mutant promoters were transfected into human embryonic kidney 293T cells along with increasing amounts of an SREBP-2 expression vector, as described in the Experimental Procedures. The negative and positive control promoters analyzed in parallel are shown as pSynTLuc and pSynSRELuc and are described elsewhere (Dooley et al., 1998). Luciferase activities were normalized to β-galactosidase that was expressed from an internal control cotransfected cytomegalovirus β-galactosidase plasmid.
Data are represented as mean ± SEM. See also Figure S3.
Cell Metabolism  , 51-61DOI: ( /j.cmet )
Copyright © 2013 Elsevier Inc. Terms and Conditions

5. Figure 3
miR-96 and miR-182 Target Key Proteins of the SREBP Maturation Pathway
(A) A cartoon depicting key molecules and trafficking of the SREBP maturation pathway is presented along with the putative target genes for miR-96 and miR-182 as Insig and Fbxw7, respectively.
(B) Immunoblotting and qPCR analyses for Insig-2 and Fbxw7 in extracts from the livers of Ch- and LE-treated mice.
Data are represented as mean ± SEM. See also Figure S4 and Tables S1 and S2.
Cell Metabolism  , 51-61DOI: ( /j.cmet )
Copyright © 2013 Elsevier Inc. Terms and Conditions

6. Figure 4
Coordinate and Reciprocal Regulation of nSREBP-2 with miR-182 or Fbxw7, Respectively
(A and B) Mice were fed chow (N) supplemented with Ch, LE, or LE followed by Ch for 1–3 days as indicated. Equal amounts of protein from two separate mice were analyzed for nSREBP-2, Fbxw7, and control proteins, as indicated by immunoblotting (A), and results were quantified and plotted as relative expression (B) along with the relative expression of miR-182 analyzed by qPCR.
(C) Hepatic protein from three individual mice was analyzed as in (A). Where indicated, a control anti-miRNA (A-miR-Con) or an anti-miRNA designed to target miR-182 (A-miR-182) were injected (30 mg/kg) on days 3, 4, and 5 of LE diet supplementation, and mice were sacrificed on day 7.
Data are represented ad mean ± SEM. See also Figure S5.
Cell Metabolism  , 51-61DOI: ( /j.cmet )
Copyright © 2013 Elsevier Inc. Terms and Conditions

7. Figure 5
Regulation of SREBPs by the miR-182 Locus through FBXW7 Is Conserved in Human Cells
(A) HeLa cells were transfected with siRNA or pre-miRNAs as indicated (10 nM, Ambion) in antibiotic-free medium as described in Materials and Methods. After 24 hr, the dishes were switched to DMEM containing 5% lipoprotein-deficient serum, 12 μg/ml Ch, and 1 μg/ml 25-hydroxycholesterol and incubated for 24 hr at 37°C. Also shown are qPCR for FBXW7 and immunoblotting for FBXW7, SREBP-1, and β-actin.
(B) The full-length FBXW7 coding sequence was cloned downstream of the constitutive RPL10 promoter with the natural FBXW7 3′UTR intact. We also prepared a version where the two predicted miR-182 targeting sites were mutated to decrease the predicted complementarity. These constructs were transfected into HeLa cells in combination with miR-182 or controls and cultured as described in the Experimental Procedures and in the figure diagram. Quantitation from a scanned image of the immunoblot is presented at the top.
Data are represented as mean ± SD. See also Figure S6.
Cell Metabolism  , 51-61DOI: ( /j.cmet )
Copyright © 2013 Elsevier Inc. Terms and Conditions

8. Figure 6
miRNA Regulation of SREBP Levels Is Accompanied by the Robust Induction of Lipid Biosynthesis
(A) HeLa cells were transfected with the indicated human pre-miRNAs alone or in combination (10 nM, Ambion), as indicated and cultured in antibiotic-free medium. After 24 hr, the dishes were switched to DMEM containing 5% lipoprotein-deficient serum with or without sterol mixture (12 μg/ml Ch and 1 μg/ml 25-hydroxycholesterol) and incubated for 24 hr at 37°C and harvested. Western blot analysis for the precursor (P) and nuclear (N) form of SREBP-1 was performed. Fatty acid synthase protein was also analyzed by immunoblotting (FASN), and β-actin was measured as a control.
(B) Companion dishes of HeLa cells were treated as in (A), and de novo synthesis of fatty acid and Ch were measured with [14C] acetate incorporation. Either control miRNA (Con) or a mixture of all three specific miRNAs (miR-96, miR-182, and miR-183) were added together. The p values for differences relative to control were ∗p = for fatty acids and ∗∗p < for Ch.
Data are represented as mean ± SEM. See also Figure S7.
Cell Metabolism  , 51-61DOI: ( /j.cmet )
Copyright © 2013 Elsevier Inc. Terms and Conditions