<p><b>A</b>) Phosphorylated STAT6 in spleens of hamsters infected with <i>L. donovani</i> determined by immunoprecipitation followed by western blot of whole splenic lysates. The samples (before immunoprecipation) were probed with anti-STAT6 anti-GAPDH antibodies to confirm equivalent protein loading. The blot shown is from tissue from a single animal, representative of 4 independent experiments. <b>B</b>) Dose-dependent induction of STAT6 activation by <i>L. donovani</i> in BHK cells transfected with a STAT6-luciferase reported vector. Data are presented as the mean and standard deviation (error bars) of the relative light units in uninfected (0 parasites) cells and cells exposed to 1–40 parasites per cell over 48 hrs of culture. Shown is data from a single experiment that is representative of 2 independent experiments. <b>C-D</b>) Phosphorylation of STAT6 in hamster peritoneal macrophages exposed to <i>L. donovani</i> (10 parasites per cell) or IL-4 (10% v/v supernatants) measured as fold-increase of mean fluorescence intensity (MFI) (<b>C</b>) or percent positive cells (<b>D</b>) by flow cytometry. Data are presented as the mean and standard deviation (error bars) and are from a single experiment that is representative of 2 independent experiments. <b>E–F</b>) Phosphorylation of STAT6 in hamster BHK cells exposed to <i>L. donovani</i> (10 parasites per cell) measured as fold-increase of mean fluorescence intensity (MFI) (<b>E</b>) or percent positive cells (<b>F</b>) by flow cytometry. Data are presented as the mean and standard deviation (error bars) and are from a single experiment that is representative of 2 independent experiments. <b>G</b>) Effect of miRNAi-mediated STAT6 knockdown on arg1 and STAT6 mRNA expression. BHK cells were transfected with a non-targeting miRNAi vector (control) or a vector specific to hamster STAT6 and the expression of arg1 and STAT6 mRNA determined by real time RT-PCR in uninfected (open bars) and 24-hr infected cells (filled bars). The data are presented as the mean and standard deviation (error bars) of the fold-increase of arg1 mRNA relative to BHK cells, determined by real time RT-PCR from a single experiment, representative of 2 independent experiments. <b>H</b>) miRNAi-mediated STAT-6 knockdown in BHK cells. The expression of STAT-6 protein in BHK cells stably transfected with a miRNAi vector targeting STAT-6 (STAT-6 KD) or transfected with a miRNAi vector coding a control sequence (Control) was determined by western blot using an anti-STAT6 antibody. An anti-GAPDH antibody was used to confirm equivalent protein loading. Data are from a single blot representative of 2 independent experiments. <b>I</b>) Effect of miRNAi-mediated STAT6 knockdown on parasite burden was determined in BHK cells that were transfected with a non-targeting miRNAi vector (control) or a vector specific to hamster STAT6. The transfected cells were infected with <i>L. donovani</i> metacyclic promastigotes and the mean and standard deviation (error bars) of the parasite burden at 4, 24, 48, and 72 hrs post-infection, determined by luminometry, is shown as data pooled from 2 independent experiments. There was no difference in parasite burden between the two groups at 4 hrs post-infection. The statistical significance of differences between groups in each of the panels is identified by asterisks (*, p<0.05; **, p<0.01; ***, p<0.001).</p
