NIH/3T3 adipocytes induced without rosiglitazone expressed more <i>Pparg</i> (PPARγ) and <i>Agt</i> (Angiotensiongen).

<p>The mRNA levels and the standard errors (n = 3) of measurement of selected genes involved in blood pressure regulation, glucose transport and lipid metabolism in NIH/3T3 adipocytes induced in the presence (+Rosi) or absence (-Rosi) of rosiglitazone were compared with 3T3-L1 adipocytes. The gene symbols used were those approved by the HUGO gene nomenclature committee (HANG) for the mouse. Asterisks on top of the graph bars indicated the difference between (+Rosi) and (-Rosi) cells was significant after Bonferroni correction for multiple comparison (p <0.0045).</p

NIH/3T3 cells formed insulin responsive adipocytes after prolonged induction.

<p>(A) The whole cell culture dish view of the oil red O stain of NIH/3T3 cells induced with (R7) or without rosiglitazone (R-7) for 7 days and regular 3T3-L1 (3T3-L1) adipocytes. (B) Oil red O stained images of NIH/3T3 cells induced without rosiglitazone for 7 (R-7) or 14 days (R-14) and with rosiglitazone for 7 days (R7). (C) The glucose uptake rate in response to insulin with the standard errors (n = 3) of measurement of cells in panel (B).</p

Comparison of PI3K/AKT signaling in NIH/3T3 and 3T3-L1 adipocytes.

<p>(A) Western blotting of phosphorylated-IRS-1(pIRS-1) and phosphorylated-insulin receptor (pIR), insulin receptor (IR), IRS-1, PDK1, PI3K,phospho-AKT (pAKT), total AKT (AKT), and β-actin in NIH/3T3 and 3T3-L1 adipocytes treated with (for 15 minutes) or without insulin. β-actin was the internal loading control. Labels were: L-Ins : 3T3-L1 -insulin, L+Ins: 3T3-L1 +insulin, N-Ins: NIH/3T3 -insulin, N+Ins: NIH/3T3 +insulin. pIRS-1 and pIR were blotted with 4G10 antibody, the rest of the proteins were probed with the antibody against the labeled protein. The blots were representative of 3 independent experiments. (B) The average band intensity normalized to beta-actin and the corresponding standard errors (error bars) were calculated after densitometry determination of each band in 3 independent blots as described in (A). None of the bands showed statistical significant difference in intensity.</p

Temporal expression profiles of the C/EBPs during differentiation of 3T3-L1 and NIH/3T3 cells.

<p>The mRNA levels and the standard errors (n = 3) of measurement of (A) C/EBPα, (B) C/EBPβ, and (C) C/EBPδ were determined by qPCR and normalized by Gapdh signal on day 0 (D0), 2 (D2) and 4(D4) in differentiation medium and day 8 in DMEM +10% FBS medium.</p

EV71 infection is blocked by dynamin-2 specific siRNA.

<p>3T3-SCARB2 cells were treated with 100 and 200 pmoles of dynamin-2 siRNA or 200 pmole of control siRNA (MOCK) prior to the infection of EV71 as following the treatment protocol as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030507#pone-0030507-g004" target="_blank">Figure 4</a> described above. After 24 or 48 hours of transfection, the detection of dynamin-2 by western blot using anti-dynamin antibody was conducted, or following the transfectants were infected with MOI = 0.04 of EV71 and incubated for 24 or 48 hours before lysate preparation. Expression of dynamin-2 in the lysates was shown in the top panel. Relative dynamin-2 protein levels in specific siRNA-treated cells compared to the levels in 48 hours of cells transfected with 200 pmoles control siRNA (Mock) as 1.0 was shown. The lower panel showed the expression of EV71 capsid protein which was detected by western blotting of MAB979 antibody. Cellular β-actin was detected by blotting the same membrane with monoclonal anti-β-actin antibody. Data represent one of two independent experiments.</p

Ad-EVVLP induction of EV71-specific IgG cross-reacts E59 and 5746 strains.

<p>Seven-week-old BALB/c mice were individually primed and boosted at 14-day intervals though oral, s.c., or i.p. routes with 10⁸ pfu Ad-EVVLP or Ad-LacZ. Serum samples collected on Day 21 were assayed for IgG against heat-inactivated (A) 5746 and (B) E59-immobilized ELISA. The results were expressed as titers for each test sample. Bars correspond to mean titers for each experimental group of 5 mice.</p

Expression of SCARB2 in RD, Vero, NIH3T3, and 3T3-SCARB2 cells.

<p>(A) Lysates prepared from the tested cells were analyzed by immunoblotting using a polyclonal rabbit anti-SCARB2 antiserum. The SCARB2 protein has molecular size of 80 KDa. The internal cellular β-actin was detected by blotting the stripped membrane with monoclonal anti-β-actin antibody which corresponded to 44 KDa products. (B) The tested cells were incubated with biotinylated anti-SCARB2 antiserum (filled curve) or without antiserum (empty curve) prior fixed by methanol and then stained with FITC-conjugated avidin. Stained cells were run on a FACScan flow cytometer and analyzed by using CellQuest software (Becton Dickinson Immunocytometry System).</p

Ad-EVVLP but not FI-EV71 protects hSCARB2-Tg mice from CVA16 challenge.

<p>One-day-old hSCARB2-Tg mice were pre-immunized twice s.c. with (A) PBS (●), 3 × 10⁷ pfu Ad-LacZ (■), or 3 × 10⁷ (▲) pfu Ad-EVVLP, or (B) 1 μg FI-EV71 vaccine (●) on Days 1 and 7 after birth prior to being challenged s.c. with 5 × 10⁵ pfu CVA16. The survival of mice was monitored on a daily basis for 15 days. The number (N) of transgenic mice was shown. A log-rank test was used for statistical analysis.</p

EV71 infection is impacted by clathrin specific inhibitors.

<p>(A) 3T3-SCARB2 cells were pretreated with different doses of CPZ for one hour prior to the addition of MOI = 0.04 of EV71. (B) 50 or 100 pmoles of siRNA specific to clathrin or 100 pmoles of control siRNA (MOCK) were transfected to 3T3-SCARB2 cells as following the treatment protocol as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030507#pone-0030507-g004" target="_blank">Figure 4</a> described above. After 24 or 48 hours of transfection, cells were following lysed and detected the content of clathrin light chain B (CLTB) by Western blot using anti-clathrin antibody. Relative expression of siRNA-targeting genes shown in the top panel represented the relative CLTB protein levels of the cells transfected with specific siRNA, compared to the expression in 48 hours transfection of the cells with 100 pmoles control siRNA (Mock) as 1.0 was shown. Lysates were also prepared after 24 or 48 hours of EV71 infection and subjected to analyze the expression of synthetic EV71 capsid protein by western blot using MAB979 antibody. The result was shown in the lower panel. Cellular β-actin was detected by blotting the same membrane with monoclonal anti-β-actin antibody. Data represent one of two independent experiments.</p

Induction of 3C-specific antibody and CD4⁺ and CD8⁺ T-cell responses in Ad-EVVLP-immunized mice.

<p>BALB/c mice were individually primed and boosted at an interval of 14 days s.c. with PBS, 10⁸ pfu Ad-EVVLP or Ad-LacZ, or 0.1 μg FI-EV71. Mice were sacrificed, and serum and splenocytes were collected on Day 21. (A) Serum was assayed for IgG against recombinant 3C-immobilized ELISA. The results are expressed as titers for each test sample. Bars correspond to the mean titers for each experimental group. Splenocytes were cultured in the presence or absence of 1.4 μg recombinant 3C protein for 48 h. (B) The proliferation of CD4⁺ T cells in response to 3C was analyzed by flow cytometry with PE-Cy5 antibodies against CD4. (C) Activated CD8⁺ T cells in splenocytes were stained with PE-Cy5-conjugated anti-CD8 antibodies and subsequently fixed and stained with PE-conjugated anti-IFN-ɣ antibody, and then analyzed using flow cytometry. The results were presented as the mean percentage of CD4⁺ or CD8⁺ T cells after antigen stimulation, compared to gated CD4⁺ T cells without antigen stimulation that was set at 0%. Five mice per group were assayed.</p