Figure 7.

IL-4 stimulation of unpolarised S.tm-infected BMDM does not affect cellular viability, radical formation and expression of radical detoxifying enzymes. Unpolarised BMDM were infected with S.tm for 1 h. Afterwards, cells were stimulated (post-stimulation) with 10 ng/mL IL-4, 100 ng/mL IFNγ or left unstimulated as indicated. (a) Determination of cell viability by analysing LDH activity after 4 h post-stimulation. (b) Phoxp47 transcript levels due to infection and 4 h post-stimulation were determined by quantitative real-time PCR and normalized to Hypoxanthine phosphoribosyltransferase (Hprt) mRNA levels using the ΔΔCT method. Data were normalised to the infected Ctrl. (c) Western blot analysis of SOD1, NRF2 and β-ACTIN expression after 14 h. (d) Quantification of ROS formation using the CellROX^TM reagent, during a 1 h infection period (left) and during a gentamicin neutralisation assay and post-stimulation over the course of 24 h. Time course data were normalised to the levels of the 30 min unstimulated uninfected Ctrl. Statistical significance was determined by one-way ANOVA with Tukey post hoc test for three groups and two-way ANOVA with Tukey post hoc tests for more groups: *** p-value < 0.001. Representative data (mean ± SEM) from two to three independent experiments with two technical replications are shown.