AG490 Prevents and Reverses Diabetes Figure 6. AG490 modulates phenotype and function of DC. A - Dendritic cells were generated from bone marrow of NOD mice treated either with AG490 or DMSO as described in materials and methods and stained directly with CD11c, MHC II, CD80 and CD86. Data shown represents CD11c gated population of pool fresh purified immature CD11c of AG490 and DMSO treated mice. B- Histograms represent expression of CD11c, CD86, CD80 and CD62L on gated CD11c population of mice treated with AG490 or DMSO in vivo. C- Bone marrow cells were isolated from NOD mice treated with AG490/DMSO for 5 weeks and then differentiated into DC in vitro as described. Total RNA (200 ng) of DC was converted into cDNA and then expression of Tgf-b1 was measured by Real-Time TaqMan Gene expression assays (Applied Biosystems). Data represents the mean (fold change) of two real-time RT-PCR experiments and the bars represent standard deviation of the mean. Fold change of Tgf-b1expression is shown (n = 5 mice/ group, * p = 0.02). D- BMDCs were generated from BALB/c mice treated in vitro with 20 mM AG490 or DMSO for 12 hrs and then co-cultured with constant numbers of CD4+CD252 T-cells (16105) with soluble anti-CD3 (2.5 ng/ml) for 72 hrs. Incorporation of 3H-thymidine was measured in the last 16 hr of cell culture. Experiment was performed in triplicate format and repeated at least twice. Data analysis was performed using the student t- test and p value#0.05 considered significant. Bars represent deviation of the mean (* p = 0.002, **p = 0.0002; ***p = 0.001). [Figures A & B are intended to be in color online and black and white in print]. doi:10.1371/journal.pone.0036079.g006 monitored at least twice a week starting at week 6 and continued thereafter. Blood glucose concentrations were determined using ACCU-CHEK Comfort Curve (Roche, Indianapolis), and diabe- tes diagnosed if values were greater than 250 mg/dl in two consecutive tests with 24 hours apart. PLoS ONE | www.plosone.org 9 May 2012 | Volume 7 | Issue 5 | e36079