Figure 5

Giardia up-regulates flavohemoglobin upon nitric oxide (NO) stress. Human intestinal epithelial cells (HCT-8) were stimulated for NO production by addition of cytokines (TNF-α (200 ng/mL), IL-1α (200 ng/mL), IFN-γ (500 ng/mL)). Giardia trophozoites of the isolate WB were added to the NO-producing host cells and to control cells after 40 h. Samples were measured for expression of the NO-detoxifying protein flavohemoglobin (FlHb) at indicated time points. A, Upon interaction with NO-producing cells FlHb was induced in trophozoites on the RNA level compared to the control gene GL50803_17364 as assessed by qPCR in technical quadruplicates. This highly significant difference is indicated by asterisks. B, Western blot detecting the expression of FlHb and the control protein Tat1 in Giardia upon interaction with HCT-8 cells with and without NO-induction. C, Quantification of the Western blot bands (B) by image J software clearly shows the induction of FlHb protein in Giardia trophozoites upon interaction with NO-induced host cells. The results are representative for similar results obtained by three independent experiments.