Figure 5
Expression of GATA-3 and SOCS-1 in infected macrophages. (A,B) Array analysis. Bone marrow-derived macrophages from CBA mice were infected for 7 days with wild-type or transfected amastigotes at a ratio of 10 parasites per macrophage. After the period of infection total RNA was extracted and used as a template to synthesise radiolabelled probes. The mouse cytokine expression array (v.1.0) from R & D Systems, on an 8 cm × 12 cm filter [56], was probed with normal macrophage cDNA, wild-type L. mexicana-infected macrophage cDNA, and cDNA from cells infected with alt-1 or alt-2 transfected L. mexicana. Quantification was carried out using a PhosphorImager and data were analyzed with ImageQuant v1.2. Results from two independent experiments are presented in adjacent bars. (C-F) Real-time RT-PCR analysis. Total RNA from infected and non-infected macrophages was extracted and single-stranded cDNA was synthesised. Relative quantification of the expression of the genes of interest was measured by real-time PCR using the LightCycler. The abundance of GATA-3 and SOCS-1 after 24 hours (C and D, data from n = 4 experiments), and after 7 days (E, n = 7, and F, n = 3) was expressed as a ratio of amplified product to the control, mouse S29 ribosomal protein. Each bar represents mean ± standard error of the mean. One-way analysis of variance showed that for GATA-3, both alt-1 and alt-2 transfectants were significantly higher than wild-type (p < 0.05) at both time points, and that for SOCS-1, both were significantly higher than wild-type (p < 0.01) at day 7.