Transient transfection of 3T3-L1/Tet-On clone by pTRE-Tight-Luc and luciferase assay.

<p>3T3-L1-pTet-On clone was transfected with pTRE-Tight-Luc plasmid. After 48 h post transfection, cells were treated with 0, 100, 1,000 ng/mL or 10,000 ng/mL of Dox and 6 h, 12 h and 48 h post treatment, the luciferase activity was analyzed using the luciferase assay system kit.</p

E4orf1 fold expression relative to pTRE.

<p>The pTRE TIGHT-E4orf1 clone was treated with 0 and 1,000 ng/mL Dox and RNA was harvested at either 16, 24 or 48 h post treatment. E4orf1 fold expression was determined relative to un-induced pTRE TIGHT-E4orf1 at 24 h using real time PCR assay.</p

Effect of regulated <i>E4orf1</i> expression on glucose disposal.

<p>3T3-E4 inducible clone and pTRE empty vector clones were induced with 1,000 ng/mL Dox for 24, 48, 72 and 96 h. After 96 h of induction, Dox was removed for 24 h or 48 h and reintroduced for 24 h or 48 h. At all-time points the glucose uptake in E4orf1 groups was significantly greater compared to the respective pTRE groups as determined by student T-Test (p<0.00001). Mean + SD. The difference of glucose uptake between pTRE and E4orf1 groups is presented, which was calculated by subtracting the average of the pTRE group values from the individual biological replicate value for the respective E4orf1 group.</p

Stable expression of 3T3-L1/pTet-On and pTRE-Tight-E4orf1 plasmids and E4orf protein expression.

<p><b>A</b>) Flourescence micrographs (40×) of live <i>E4orf1</i> double stable inducible clone induced with 1,000 ng/mL doxycycline for 24 h. Phase, p-Tet-On showing green fluorescence, p-TRE-Tight-E4orf1-Red fluorescence and Overlay. Arrows indicate co-localization. B) Western blot showing E4orf1 protein expression in pTRE and 3T3-E4 clone.</p

Two bar graphs showing the time spent in different parts of the open field and elevated plus maze, respectively, of the three groups of mice.

<p>No significant differences between the groups were present.</p

Two series of coronal sections of the dorsal hippocampus stained for Aβ.

<p>A non-significant decrease in Aβ staining in stratum oriens and a significant reduction in Aβ staining in the dentate gyrus in both LY and CR mice compared to the control is shown. Lower panel, Aβ levels measured by ELISA. Insoluble Aβ-40 levels are significantly reduced in CR and LY groups compared to the control (*student's t-test p = 0.03 for both comparisons). Insoluble Aβ-42 levels are reduced in CR and LY compared to the control (**Tukey-Kramer HSD p = 0.02 for both comparisons). so - stratum oriens; sp - stratum pyramidale.</p

E4orf1 requires its PBM for glucose uptake and Ras activation.

<p>3T3-L1 cells were transfected with V-5 tagged plasmids expressing E4orf1 (pcDNA-V5-AD36-E4orf1), mutant E4orf1 (pcDNA-V5- AD36-E4ORF1ΔPBM) or a null vector (pcDNA-V5-DEST) and a Ras activation assay was conducted the next day. The activated slurry and 3% of whole cell lystate was loaded to SDS-PAGE and probed for Ras. Also 3T3-L1 cells that constitutively express Ad36 E4orf1, a mutated E4orf1 (deleted PBM; ΔPBM) or Null vector were plated in 12 well plates and a 2DG uptake assay was conducted the following day. <b>A</b>) WB for Ras activation assay: pcDNA-V5-AD36-E4orf1 activates Ras (p = 0.04), whereas the Null vector (pcDNA-V5-DEST) does not. pcDNA-V5- AD36-E4ORF1ΔPBM did not activate Ras, suggesting E4orf1 depends on its PBM region for Ras activation. Densitometry expressed as ratio of activated Ras to normalized total Ras from 3% WCL WB. <b>B</b>) 2DG uptake in constitutive expressing Ad36 E4orf1, a mutated E4orf1 (deleted PBM; ΔPBM) or Null vector normalized to protein: E4orf1 increased glucose uptake compared to Null vector (p = 0.008), and there was no difference in glucose uptake when the PBM region of E4orf1 is mutated.</p

Four bar graphs showing the data from the inflammation measurements, top row, density measurements of Iba1 and GFAP staining in stratum oriens of the hippocampus, respectively (p = 0.0042 for Iba-1).

<p>Bottom row, ELISA measurements of IL-6 and IL-10, respectively, levels in the hippocampal formation.</p

E4orf1 interacts with Dlg1 and activates Ras.

<p>HEK293 were co-transfected with GW1-CMV-HA-Dlg1 and either pcDNA-V5-Ad36 E4orf1 or a control plasmid (pcDNA-V5-DEST), and a pull down for HA was conducted and loaded to SDS-PAGE. WB were probed with V5 antibody to detect Dlg1 and E4orf1 interaction. Also, 3T3-E4 and pTRE Null cell lines were treated with 1,000 ng/ml Doxycycline, and a Ras activation assay was conducted. Following pull down, activated slurry and 6% of the whole cell lysate were loaded to SDS-PAGE and probed with total Ras antibodies, or H-, N-, or K-Ras specific antibodies. Blots were also probed with β-actin, and densitometry was expressed as ratio of activated Ras to normalized total Ras. <b>A</b>) WB show HA-Dlg1 and V5E4orf1 in the same complex after IP, indicating the binding of Ad36 E4orf1 with Dlg1. WB also shows the presence of Dlg1 in the whole cell lysate (WCL). As expected, V5-E4orf1 is absent in pcDNA-V5-DEST group after IP for HA-Dlg1. <b>B</b>. (i) WB for total, H-, and N-Ras activation. T = total (6% WCL) and A = Activated slurry. (ii) Total Ras abundance normalized to β-actin was significantly more in 3T3-E4 compared to pTRE Null vector cell line (p = 0.04). (iii)Total Ras activation densitometry, expressed as ratio of activated to normalized total Ras. Total Ras activation was significantly greater in 3T3-E4 compared to pTRE Null (p = 0.01). (iv) H-ras activation densitometry, expressed as ratio of activated H-Ras to normalized total H-Ras. H-Ras activation was significantly more in 3T3-E4 compared to pTRE Null (p = 0.01).</p

Left: Change in body weight over the treatment period.

<p>Note that the control and LY animals gain equal weight during the treatment period, but the CR animals are significantly lighter; further, the CR mice had a significantly (p<0.001) lower fat mass compared to the other two groups. Right: Bar graph showing the percent of fat of the total body weight as determined by QMR. Note the reduced fat content of the CR mice.</p