1. Volume 17, Issue 12, Pages 3305-3318 (December 2016)
Adenovirus Protein E4-ORF1 Activation of PI3 Kinase Reveals Differential Regulation of Downstream Effector Pathways in Adipocytes 
Natasha Chaudhary, Eva Gonzalez, Sung-Hee Chang, Fuqiang Geng, Shahin Rafii, Nasser K. Altorki, Timothy E. McGraw 
Cell Reports 
Volume 17, Issue 12, Pages (December 2016)
DOI: /j.celrep
Copyright © 2016 The Author(s) Terms and Conditions

2. Cell Reports 2016 17, 3305-3318DOI: (10.1016/j.celrep.2016.11.082)
Copyright © 2016 The Author(s) Terms and Conditions

3. Figure 1
Ad5 E4-ORF1 Expression Enhances Growth-Factor-Stimulated Glut4 Translocation without an Effect on General Endocytic Trafficking
(A) qRT-PCR showing relative mRNA expression of E4-ORF1 in control and stably E4-ORF1-expressing adipocytes.
(B) Representative western blot of whole-cell lysates from control and stably FLAG-E4-ORF1-expressing adipocytes probed for FLAG.
(C) Anti-Flag IF for transiently expressing (left) and stably FLAG-E4-ORF1 (right)-expressing adipocytes. Epifluorescence (EPI) images, anti-Flag staining in green, and nuclei (DAPI) in red are shown. Arrows indicate cells with prominent plasma membrane anti-Flag staining. Scale bar, 20 μm.
(D) Schematic of Glut4 reporter depicting the location of the tags.
(E and F) Quantification of surface (Cy3 anti-HA fluorescence)-to-total (GFP fluorescence) ratio of HA-Glut4-GFP expression in transient (E) and stable (F) E4-ORF1-expressing adipocytes in basal and insulin-treated condition. Data are normalized to the HA-Glut4-GFP surface-to-total value for control cells with 1 nM insulin treatment for each experiment. n = 6 assays.
(G) Representative Airyscan confocal images showing GFP expression for HA-Glut4-GFP and endogenous Syntaxin 6 staining (Cy3) in basal and 0.1 nM insulin-treated control and E4-ORF1-expressing adipocytes. Scale bar, 5 μm.
(H) Quantification of surface-to-total ratio of HA-Glut4-GFP in control, control adipocytes expressing FLAG-E4-ORF6, E4-ORF1 stable adipocytes, and E4-ORF1 stable adipocytes co-expressing FLAG-E4-ORF6, respectively, under basal and insulin-treated conditions. n = 3 assays.
(I) Quantification of surface-to-total ratio of HA-Glut4-GFP expression in control and stable E4-ORF1 adipocytes under basal, insulin, and IGF-treated conditions. Data are normalized to the HA-Glut4-GFP surface-to-total value for insulin-treated control cells for each experiment. n = 3 assays.
(J) Quantification of surface-to-total ratio of transferrin receptor in control and E4-ORF1-expressing adipocytes under basal and insulin-treated conditions. Data are normalized to the HA-Glut-GFP surface-to-total value for 1 nM insulin-treated control cells for each experiment. n = 3 assays. Data represent mean normalized values ±SEM. ∗p < 0.05, ∗∗p < See also Figure S1.
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4. Figure 2 E4-ORF1 Activates Akt
Control and stably expressing E4-ORF1 adipocytes were treated with indicated insulin concentrations, and the lysates were subjected to western blot analysis. Representative blot showing (A) phospho-Akt S473, (B) phospho-Akt T308 and total Akt expression (upper) and respective densitometric analysis of immunoblots (lower), (C) phospho-AS160 T642 and total AS160 expression (upper) and densitometric analysis of immunoblots (lower), and (D) phospho-FoxO1 S256 and total FoxO1 expression (upper) and densitometric analysis of immunoblots (lower). For densitometric analysis, n = 3 assays. Mean values ±SEM, ∗p < 0.05.
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5. Figure 3
E4-ORF1 Signals through p110α and Akt and Mimics the Effects of Insulin on FoxO1
(A) Both control and stable E4-ORF1 adipocytes were transfected with FoxO1-GFP and treated with indicated insulin concentration. Data show percentage of cells with cytosolic FoxO1-GFP under basal and insulin-treated conditions. n = 4 assays.
(B) Control and stably expressing E4-ORF1 adipocytes were pre-treated with PIK75 (0.1 μM), TGX221 (0.1 μM), or with DMSO (vehicle) for 1 hr followed by insulin stimulation. Quantification of surface-to-total ratios of HA-Glut4-GFP are shown. Data are normalized to the HA-GLUT4-GFP surface-to-total value for insulin-treated control cells for each experiment. n = 4 assays.
(C) Quantification of surface-to-total ratio of HA-Glut4-GFP measured in control, transiently expressing Flag-E4-ORF1 wild-type and Flag-E4-ORF1-V128A adipocytes under basal and insulin-treated conditions. Data are normalized to the HA-Glut-GFP surface-to-total value for basal control cells for each experiment. n = 4 assays.
(D) Control, transiently expressing Flag-E4-ORF1 wild-type, and Flag-E4-ORF1-V128A adipocytes were transfected with FoxO1-GFP. Data show percentage of cells with nuclear FoxO1-GFP under basal condition. n = 3 assays.
(E) Control and adipocytes stably expressing E4-ORF1 were pre-treated with DMSO (vehicle) or MK2206 (1 μM) for 1 hr followed by insulin stimulation. Quantification of surface-to-total ratios of HA-Glut4-GFP are shown. Data are normalized to the HA-Glut4-GFP surface-to-total value for insulin-treated control cells for each experiment, n = 4 assays.
(F) FoxO1-GFP-expressing control and E4-ORF1-expressing adipocytes were treated with DMSO (vehicle), PIK75 (0.1 μM), TGX221 (0.1 μM), or with MK2206 (1 μM), respectively, for 1 hr. Data show percentage of cells with nuclear FoxO1-GFP under basal condition. n = 4 assays. Data represent mean normalized values ±SEM, ∗p < 0.05, ∗∗p < 0.005, ∗∗∗p < See also Figures S2 and S3.
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6. Figure 4 E4-ORF1 Colocalizes with p110α and Activates PI3K Activity
(A) Both control and stable E4-ORF1-expressing adipocytes were transfected with GFP-PH1. Quantification of plasma membrane (TIRF GFP measurements) to total (EPI GFP measurements) ratio of GFP-PH1 expression in basal and insulin-stimulated conditions is shown. Data are normalized to GFP-PH1 TIRF-to-EPI value for control cells under basal condition in each experiment. n = 4 assays.
(B) GFP-PH1-expressing control and stable E4-ORF1 adipocytes were treated with PIK75 (0.1 μM) for 1 hr. Quantification of TIRF to EPI ratio of GFP-PH1 expression under basal condition is shown. Data are normalized to GFP-PH1 TIRF-to-EPI value for control untreated cells in each experiment. n = 3 assays.
(C) Representative immunoblots for p110α, p85, and actin expression in control and stable E4-ORF1-expressing adipocytes in basal conditions.
(D) Representative TIRF images for FLAG-E4-ORF1 in basal and 0.1 nM insulin-stimulated conditions. Scale bar, 10 μm.
(E) Quantification of TIRF imaging shown in (D). The fluorescence power of anti-Flag staining in the TIRF zone divided by anti-Flag fluorescence staining in EPI for individual cells in basal and 0.1 nM insulin-stimulated condition. Individual points are data from single cells. n = 40 cells.
(F) Representative TIRF image showing colocalization between E4-ORF1 (anti-FLAG staining) and endogenous p110α stained puncta in basal adipocytes. Scale bar, 10 μm.
(G) Quantification of the endogenous p110α puncta that also contain E4-ORF1 (anti-FLAG staining). n = 2 assays. Mean values ±SD.
(H) Quantification of the colocalization between E4-ORF1 (anti-FLAG staining) and endogenous insulin receptor. n = 2 assays. Mean values ±SD.
Data represent mean normalized values ±SEM unless otherwise stated, ∗p < 0.05, ∗∗p < See also Figure S4.
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7. Figure 5
E4-ORF1 Enhances Glut4 Translocation to the Plasma Membrane by Increasing Glut4 Exocytosis Independent of Rab10
(A) qRT-PCR showing relative mRNA expression of Rab10 in control adipocytes and stable Rab10 knockdown (KD) adipocytes.
(B) Quantification of surface-to-total ratio of HA-Glut4-GFP in basal and insulin-stimulated conditions performed in Rab10 KD adipocytes ectopically re-expressing Rab10 (control), Rab10 KD adipocytes ectopically co-expressing Rab10 and Flag-E4-ORF1 (E4-ORF1), Rab10 KD adipocytes, and Rab10 KD adipocytes expressing Flag-E4-ORF1, respectively. Data are normalized to the HA-Glut4-GFP surface-to-total value for insulin-treated control cells for each experiment. n = 4 assays.
(C) Average insulin-stimulated exocytic rate constant for HA-Glut4-GFP measured in control and stable E4-ORF1 adipocytes. n = 5 assays.
(D) Schematic of method to measure HA-Glut4-GFP accumulation in the TIRF zone (evanescent field) (TIRF-GFP/EPI-GFP fluorescence ratio) and the fraction of Glut4 that is inserted into the PM (anti-HA TIRF-Cy3/TIRF-GFP fluorescence ratio).
(E) Representative TIRF images for HA-Glut4-GFP, showing anti-HA TIRF-Cy3 fluorescence and TIRF-GFP fluorescence in control and stable E4-ORF1 adipocytes under basal and 0.1 nM insulin-stimulated conditions. Scale bar, 10 μm.
(F) Quantification of the fraction of Glut4 inserted into the PM by measuring anti-HA TIRF-Cy3 fluorescence to TIRF-GFP fluorescence ratio, in control and stable E4-ORF1 adipocytes under basal and insulin stimulated conditions. Data are normalized to the TIRF-Cy3-to-TIRF-GFP value for insulin-treated control cells for each experiment. n = 3 assays. Data represent mean normalized values ±SEM, ∗p < 0.05.
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8. Figure 6
E4-ORF1 Increases Glucose Uptake by Increasing Glut1 in the Plasma Membrane of Adipocytes
(A) 3H-2-Deoxyglucose uptake, normalized to amount of protein in each sample, in control and stable E4-ORF1 adipocytes in basal and insulin-stimulated conditions. Data are normalized to uptake for insulin-treated control adipocytes for each experiment. n = 5 assays.
(B) 3H-2-Deoxyglucose uptake, normalized to amount of protein in each sample, in control and stable E4-ORF1 adipocytes either treated with MK2006 or vehicle control (DMSO), under unstimulated conditions. n = 3 assays.
(C) Representative immunoblots for Glut4, Glut1, and respective actin expression in control and stable E4-ORF1 adipocytes cell lysate.
(D) Quantification of surface-to-total ratio of HA-Glut1 expression in control and E4-ORF1-expressing adipocytes under basal alone, pre-treatment with PIK75 and MK2206, respectively. n = 4 assays.
(E) Quantification of surface-to-total ratio of HA-Glut1 expression in Rab10 KD adipocytes ectopically re-expressing Rab10 (control), Rab10 KD adipocytes ectopically co-expressing Rab10 and Flag-E4-ORF1 (E4-ORF1), Rab10 KD adipocytes, and Rab10 KD adipocytes expressing Flag-E4-ORF1, respectively, under basal conditions. E4-ORF1 data were normalized to Glut1 surface-to-total amount in control adipocytes, and Rab10 KD-expressing E4-ORF1 data were normalized to Glut1 surface-to-total amount in Rab10 KD adipocytes for each experiment. n = 4 assays.
(F) ELISA-based quantification of lactate in media of control, stable E4-ORF1 adipocytes, and stable E4-ORF1 adipocytes with Glut1 KD. Data were normalized to lactate concentration in control adipocytes for each experiment. n = 3 assays. Mean normalized values ±SD.
(G) Mean-difference plot representing differential gene expression of 2,136 genes. Genes colored in red represent differentially expressed genes (adjusted p < 0.05) in stable E4-ORF1 adipocytes compared to control cells. n = 3 per group.
(H) Heatmap of differentially expressed genes in control and stable E4-ORF1 adipocytes with more than 2-fold change in expression, color coded on basis of fold change relative to control adipocytes (290 total genes; green, 153 downregulated genes; red, 137 upregulated genes), n = 3 per group, adjusted p value <0.05.
(I) Quantification of relative mRNA expression levels of listed genes from differentially expressed gene set of E4-ORF1 adipocytes. Data represent mean normalized values ±SEM unless otherwise stated, ∗p < 0.05, ∗∗p < 0.005, ∗∗∗p < 0.001, ∗∗∗∗p < See also Figure S5 and Tables S1 and S2.
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