1. Volume 3, Issue 6, Pages 1847-1856 (June 2013)
Nontelomeric Role for Rap1 in Regulating Metabolism and Protecting against Obesity 
Frank Yeung, Cristina M. Ramírez, Pedro A. Mateos-Gomez, Alexandra Pinzaru, Giovanni Ceccarini, Shaheen Kabir, Carlos Fernández- Hernando, Agnel Sfeir 
Cell Reports 
Volume 3, Issue 6, Pages (June 2013)
DOI: /j.celrep
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2. Cell Reports 2013 3, 1847-1856DOI: (10.1016/j.celrep.2013.05.032)
Copyright © 2013 The Authors Terms and Conditions

3. Figure 1 Obese Phenotypes in Rap1-Deficient Mice
(A) Rap1-KO mice display morphological signs of obesity when compared to Rap1-WT controls. Shown are representative photographs of 24-week-old female (left) and male (right) mice.
(B) Body-weight curves of Rap1-WT (white boxes) and Rap-KO (black circles) mice over a period of 56 weeks. Animals were fed a normal chow diet and weighed once every 2 weeks. Data represent mean value, and p values were calculated with the Student’s t test (∗p < 0.05).
(C) Plasma glucose levels during glucose tolerance test (GTT) performed on 6- to 8-week-old female mice. Closed circles represent Rap1-KO mice (n = 5) and open boxes represent WT control mice (n = 5). Values were normalized to t = 0 time point. Data represent mean ±SEM. All p values were calculated with the Student’s t test (∗p < 0.05).
(D) Plasma glucose levels during Insulin tolerance test (ITT) performed on 6-8 week-old female mice (n = 5 for each group). p values were calculated by Student’s t test and (∗) indicates p < 0.05.
(E) GTT performed on 24-32 week-old females (right panel) and males (left panel) mice. Closed circles represent Rap1-KO mice (n = 15 for females and n = 14 for males) and open boxes represent WT control mice (n = 18 for females and n = 20 for males). Data represent mean ±SEM. All p values were calculated with the Student’s t test (∗p < 0.05).
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4. Figure 2 White Adipose Tissue Dysregulation in the Absence of Rap1
(A) Representative photograph of 40-week-old Rap1-KO (left) and Rap1-WT (right) female mice displaying WAT accumulation.
(B) A DEXA scan was performed on 24- to 32-week-old Rap1-WT mice. Fat percentage is represented as average values ±SEM. The Student’s t test was performed for significance and (∗p < 0.05).
(C) Shown are representative sections of intra-abdominal WAT from 6-week-old Rap1-WT and Rap1-KO mice stained with H&E. Morphometric analysis of white fat cells was carried out using MetaMorph. Cell area was quantified for each genotype and values represent mean ±SEM.
(D) Gene Ontology analysis was performed to classify differentially expressed genes from intra-abdominal WAT of 6-week-old Rap1-KO and Rap1-WT female mice into biological processes.
(E) Transdifferentiation of MEFs isolated from Rap1-KO and Rap1-WT mice into adipocytes. Lipid accumulation is visualized using oil red O staining.
(F) Shown is a representative image of a transdifferentiated adipocyte stained with oil red O. Lipid accumulation was quantified and normalized to WT levels. Values are mean ±SEM. A Student’s t test was performed for significance and (∗p < 0.05).
Cell Reports 2013 3, DOI: ( /j.celrep )
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5. Figure 3 Rap1 Deficiency Alters Liver Metabolism, Leading to Steatosis
(A) Shown is a representative photograph of 24-week-old Rap1-KO and Rap1-WT mice, displaying pale/fatty liver (marked by asterisk) in the absence of Rap1.
(B) Representative H&E (upper) and oil red O (bottom) staining liver sections from 20-week-old WT and Rap1-KO mice. The red stain is an indicator for accumulation of neutral lipids.
(C) Microarray analysis of messenger RNA from the livers of 6-week-old Rap1-KO and Rap1-WT female mice. Heat map representation of differentially expressed metabolism and inflammation genes. Average gene expression is colored proportionally to signal intensity with blue denoting a lower expression and red representing high expression.
(D) Gene Ontology analysis and classification of differentially expressed genes into biological processes.
(E) A Venn diagram showing the overlap of Rap1-regulated genes (red) with the PPARα pathway (blue) genes identified using GSEA analysis (p = ; Fisher’s exact test).
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6. Figure 4
Rap1ΔTRF2 Complements Transcriptional Defects of Rap1 Null Cells
(A) Schematic of mouse Rap1 protein with its different domains: BRCT, Myb, and RCT. Highlighted in red is the amino acid residue that is important for TRF2 interaction and that was mutated in the Rap1ΔTRF2 allele.
(B) Coimmunoprecipitation of TRF2 from 293T cells cotransfected with wild-type Rap1 and mutant Rap1ΔTRF2.
(C) Immunofluorescence showing the localization of WT Rap1 and Rap1ΔTRF2 allele (detected with MYC antibody in green) in Rap1-deficient MEFs. Telomeres are detected with a PNA probe in red and DNA is counterstained with DAPI.
(D) Quantification of lipid accumulation in adipocytes derived from Rap1-KO MEFs expressing vector control, WT Rap1, and Rap1ΔTRF2. Cells were stained with oil red O 8 days after hormonal induction and levels of lipid were expressed as percentage relative to adipocytes originating from cells expressing Rap1-WT. Values are mean ±SEM. A Student’s t test was performed for significance (∗p < 0.05).
(E) Global transcriptional analysis of Rap1-KO MEFs complemented with vector control, WT Rap1, and Rap1ΔTRF2. Shown on the left is a row-centered heat map with hierarchical clustering carried out on the 187 differentially expressed genes. Represented on the right are examples of differentially expressed genes rescued by the expression of Rap1ΔTRF2.
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7. Figure S1
Altered Metabolism in Rap1 Deficient Mice, Related to Results
(A) Genotyping PCR of tail DNA of mice with the indicated genotype. See materials and methods for primer sequence.
(B) Western blot for Rap1 protein in MEF with the indicated genotype.
(C) Daily food intake of Rap1 WT (gray bars; (n = 12 for females and n = 14 for males) and KO (black bars; n = 20 for females and n = 22 for males) mice. Chow mass is represented as average values ± SEM.
(D) Blood glucose analysis performed by glucometer from blood collected from tail vein of 20-32 week old mice after overnight fasting. Rap1-WT n = 22, gray bar. Rap1-KO: n = 15, black bar.
(E–G) Plasma insulin, triglycerides, and cholesterol levels from Rap1 WT (gray bars) and Rap1-KO (black bars) mice, 20-32 week-old. Data represented as average values ± SEM. Student’s t test was performed for significance and ∗ indicates p < No significant difference was noted in triglyceride levels between the two cohorts of mice.
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8. Figure S2
Rap1 Null Mice Display No Telomere Dysfunction, Related to Results
(A) Immunohistochemistry for the detection of γ-H2AX in liver, skin epidermis, and kidney sections of Rap1-WT and Rap1-KO mice (56 week-old). Tissue section derived from an irradiated (5 Gy) mouse serve as positive control.
(B) Immunoblot for CHK2 in adipose tissue and cell extracts of Rap1-WT and Rap1-KO mice (56 week-old). Positive Control sample represent IR treated cells (1 hr after 2-Gy dose).
Cell Reports 2013 3, DOI: ( /j.celrep )
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9. Figure S3 No Thermogenic Defect in Rap1 Null Mice, Related to Results
(A) H&E staining of paraffin-embedded brown fat sections from Rap1-KO and Rap1-WT 6 week-old mice.
(B) Analysis of thermogenesis in Rap1-KO mice. Graph represents time course body temperature recordings of 6-7 week-old Rap1-WT (circle, n = 5), and Rap1-KO (square, n = 6) mice exposed to cold temperature (4°C). Temperatures were measured hourly with a rectal thermometer. Shown is the mean ± SEM. Student’s t test was performed for significance and (∗) denotes p < (∗∗) denotes p < 0.01.
(C) Quantitative real-time PCR analysis for Ucp1 gene expression in brown fat. Interscapular brown fat was dissected from Rap1-WT and Rap1-KO mice maintained at 24°C or exposed to 4°C for 5 hr. Primers specific for β-actin were used for normalization. Ucp1 gene expression is represented relative to the room temperature control. The mean ± SEM is shown.
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10. Figure S4
qRT-PCR Validation of Deregulated Genes in Rap1-KO versus WT Liver, Related to Results
Gene expression is represented with respect to wild-type sample. The analysis was done in triplicate. Shown is the mean ± SEM. Student’s t test was performed for significance and (∗) denotes p < 0.05.
Cell Reports 2013 3, DOI: ( /j.celrep )
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11. Figure S5
Rap1ΔTRF2 Complementation Is Correlated with Rap1ΔTRF2 Recruitment to Differentially Expressed Genes, Related to Results
Schematic of differentially expressed genes rescued in Rap1-KO mouse embryonic fibroblasts expressing Rap1ΔTRF2. Rap1 bound genes were determined by association of ChIP-seq peak data with mouse genomic annotations. ChIP-seq binding sequences were scanned for permutations of the TTAGGG motif allowing for one mismatch. Fisher’s exact test was performed for significance and (∗) indicates p < 0.05.
Cell Reports 2013 3, DOI: ( /j.celrep )
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12. Figure S6
Expression Levels and TIF Analysis of Rap1 and Rap1ΔTRF2 in Rap1-KO MEFs, Related to Results
(A) Western blot analysis using anti-Myc antibody for Rap1-KO MEFs infected with Myc-tagged WT or mutant Rap1.
(B) Western blot using anti-Rap1 antibody of Rap1-WT MEFs infected with wild-type or mutant Rap1. MEFs infected with diluted virus titers show expression levels of both WT Rap1 and Rap1ΔTRF2 comparable to endogenous levels of Rap1 in control MEFs.
(C) TIF analysis for Rap1-KO MEFs infected with vector control and Rap1ΔTRF2. Representative images of cells stained with 53BP1 antibody in green. Telomeres were labeled in red and nuclei were counterstained in DAPI.
Cell Reports 2013 3, DOI: ( /j.celrep )
Copyright © 2013 The Authors Terms and Conditions